Co-administration of an agent linked to an internalization peptide with an anti-inflammatory

ABSTRACT

The invention provides methods of delivering pharmacologic agents linked to an internalization peptide, in which an inflammatory response inducible by the internalization peptide is inhibited by co-administration of an anti-inflammatory or by linking the internalization peptide to biotin or similar molecule. Such methods are premised in part on the results described in the examples whereby administration of a pharmacological agent linked to tat at high dosages is closely followed by an inflammatory response, which includes mast cell degranulation, histamine release and the typical sequelae of histamine release, such as redness, heat, swelling, and hypotension.

This application claims priority from U.S. Provisional App. No.61/185,943, filed Jun. 10, 2009, and is a continuation in part of U.S.application Ser. No. 12/323,915 filed Nov. 26, 2008, which claimspriority to U.S. Provisional App. No. 60/992,678 filed Dec. 5, 2007,each incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED IN A COMPUTER READABLE FORMAT

The sequence listing written in the file 413219_SEQLST.TXT is 18,027bytes, and was created on Jun. 11, 2012 and is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

Many drugs are required to be taken up by cells or pass through cellsand/or be taken up by cellular organelles to reach their intendedtherapeutic target. Many larger molecules and some small ones bythemselves have limited capacity to pass through cellular membranes. Thecapacity to pass through cellular membranes can be increased by linkinga pharmacological agent to an internalization peptide (also known asprotein transduction domains, or membrane translocation domains). Thesepeptides include tat, antennapedia peptide and arginine-rich peptides.These peptides are short basic peptides present in many cellular andviral proteins and serve to mediate translocation across membranes. Acommon feature of these peptides is their highly cationic nature. Suchpeptides have been reported to facilitate uptake of many differentpeptide and proteins into cells, as well as oligonucleotides, peptidenucleic acids and small molecules and nanoparticles. Uptake into cellsand organelles and across the blood brain barrier has been reported.

As one application of internalization peptides, a tat peptide has beenlinked to a peptide inhibitor of interaction between postsynapticdensity-95 protein (PSD-95) and NMDARs (Aarts et al., Science 298,846-850 (2002)). The resulting chimeric peptide was tested in a cellularand an animal model of stroke. The chimeric peptide was taken up intoneuronal cells and found to reduce ischemic brain damage in the animalmodel. This result has led to the proposal to use peptide antagonists ofPSD-95/NMDAR linked to an internalization peptide for treating strokeand other diseases mediated by excitotoxicity.

SUMMARY OF THE INVENTION

The invention provides a method of inhibiting cerebral ischemia due toendovascular surgery, comprising: administering to a subject undergoingendovascular surgery a pharmacologic agent that inhibits binding ofPSD95 to NDMAR 2B linked to an internalization peptide in a regimeeffective to inhibit cerebral ischemia; and administering to the subjecta mast cell degranulation inhibitor, whereby the mast cell degranulationinhibitor can inhibit an anti-inflammatory response inducible by theinternalization peptide and/or the mast cell degranulation inhibitor isadministered within a period of 30 minutes before to 15 minutes afterthe pharmacological agent.

The invention further provides a pharmacologic agent that inhibitsbinding of PSD95 to NDMAR 2B linked to an internalization peptide foruse in inhibiting cerebral ischemia due to endovascular surgery incombination with a mast cell degranulation inhibitor to inhibit aninflammatory response inducible by the internalization peptide.

The invention further provides a mast cell degranulation inhibitor foruse in inhibiting cerebral ischemia due to endovascular surgery incombination with a pharmacologic agent that inhibits binding of PSD95 toNDMAR 2B linked to an internalization peptide, wherein the mast celldegranulation inhibitor inhibits an inflammatory response inducible bythe internalization peptide.

Optionally, the mast cell degranulation inhibitor is administered at thesame time as or up to 15 minutes before the pharmacological agent.Optionally, the mast cell degranulation inhibitor is co-formulated withthe pharmacologic agent. Optionally, the mast cell degranulationinhibitor is administered by a peripheral route. Optionally, the mastcell degranulation inhibitor and pharmacologic agent are administeredintravenously. Optionally, the subject is suffering from an episode of adisease and the pharmacological agent and the mast cell degranulationinhibitor are administered once during the disease episode. Optionally,the administration of the mast cell degranulation inhibitor does notcomport with a recurring regime of administering the mast celldegranulation inhibitor to the patient without the pharmacologic agent.Optionally, the internalization peptide is a tat peptide. Optionally,the tat peptide has an amino acid sequence comprising RKKRRQRRR (SEQ IDNO:51) or GRKKRRQRRR (SEQ ID NO:1). Optionally, the tat peptide has anamino acid sequence comprising YGRKKRRQRRR (SEQ ID NO:2), or FGRKKRRQRRR(SEQ ID NO:3), or GRKKRRQRRRP (SEQ ID NO:4). Optionally, thepharmacologic agent is a peptide, such as KLSSIESDV (SEQ ID NO:5).

The invention further provides a method of treating or effectingprophylaxis of a disease mediated by excitotoxicity comprisingadministering to a subject having or at risk of the disease andeffective regime of a peptide having an amino acid sequence consistingof or comprising (RRRQRRKKRGYKLSSTESDV SEQ ID NO:70) to treat or effectprophylaxis of the disease. Optionally, the method further comprisesadministering a mast cell degranulation inhibitor and/or ananti-histamine.

The invention further provides a peptide having an amino acid sequenceconsisting of or comprising (RRRQRRKKRGYKLSSIESDV SEQ ID NO:70) for usein treatment or prophylaxis of disease.

The invention further provides a method of delivering a pharmacologicagent to a subject, the method comprising: administering thepharmacologic agent linked to an internalization peptide to the subject;and administering a mast cell degranulation inhibitor to the subject,whereby the lodoxamide can inhibit an inflammatory response inducible bythe internalization peptide; and the mast cell degranulation inhibitoris tranilast, lodoxamide, azelastine, bepotastine, chlorzoxazone,epinastine, isoproterenol, olopatadine, pemirolast, pimecrolimus orpirbuterol.

Optionally, the mast cell degranulation inhibitor is administered at thesame time as or up to 15 minutes before the pharmacological agent.Optionally, the mast cell degranulation inhibitor is co-formulated withthe pharmacologic agent. Optionally the method is for treating orprophylaxis of a disease mediated by excitotoxicity in the subject.Optionally, the pharmacological agent is a PL peptide of an NMDARreceptor. Optionally, the internalization peptide is a tat peptide, forexample having an amino acid sequence comprising RKKRRQRRR (SEQ IDNO:51), GRKKRRQRRR (SEQ ID NO:1), YGRKKRRQRRR (SEQ ID NO:2), FGRKKRRQRRR(SEQ ID NO:3) or GRKKRRQRRRPQ (SEQ ID NO:4).

Optionally, the disease is stroke or the subject is at risk of transientcerebral ischemic attack as a result of undergoing surgery. Optionally,the mast cell degranulation inhibitor is administered by a peripheralroute. Optionally, the mast cell degranulation inhibitor is administeredwithin a window of 30 minutes before to 15 after administering thepharmacologic agent. Optionally, the mast cell degranulation inhibitoris administered within a window of 15 minutes before to the same time asadministering the pharmacologic agent. Optionally, the subject issuffering from an episode of a disease and the pharmacological agent andthe mast cell degranulation inhibitor are administered once during thedisease episode. Optionally, the administration of the mast celldegranulation inhibitor does not comport with a recurring regime ofadministering the mast cell degranulation inhibitor to the patientwithout the pharmacologic agent. Optionally, the mast cell degranulationinhibitor and pharmacological agent linked to the internalizationpeptide are co-formulated. Optionally, the co-formulation isadministered intravenously.

The invention further provides a kit comprising a pharmacological agentlinked to an internalization peptide, and lodoxamide. The inventionfurther provides lodoxamide for use in inhibiting a mast celldegranulation response inducible by a pharmacologic agent linked to aninternalization peptide. The invention further provides a pharmacologicagent linked to an internalization peptide for use in treatment orprophylaxis of disease in combination with lodoxamide to suppress andinflammatory response inducible by the internalization peptide. Theinvention further provides a pharmacologic agent that inhibits bindingof PSD95 to NDMAR 2B linked to an internalization peptide in a regimeeffective to treat or effect prophylaxis of a diseases mediated byexcitotoxicity in combination with lodoxamide to inhibit an inflammatoryresponse inducible by the internalization peptide. The invention furtherprovides lodoxamide for use in treatment or prophylaxis of a diseasemediated by excitotoxicity in combination with a pharmacologic agentthat inhibits binding of PSD95 to NDMAR 2B linked to an internalizationpeptide, wherein the lodoxamide inhibits an inflammatory responseinducible by the internalization peptide. The invention further providesco-formulation comprising lodoxamide and a peptide having an amino acidsequence of SEQ ID NO:6 (YGRKKRRQRRRKLSSIESDV) and water. Optionally,less than 5% by weight of the lodoxamide and less than 5% by weight ofthe peptide is in particulate form. Optionally, the co-formulationfurther comprises sodium chloride at a concentration of 50-200 mM.Optionally, the concentration of lodoxamide is 0.5-1 mg/ml and theconcentration of the peptide is 5-20 mg/ml.

The invention further provides a method of delivering a pharmacologicalagent linked to an internalization peptide to a population of subjectshaving or a risk of a disease treatable by the pharmacological agent,comprising administering the pharmacological agent linked to theinternalization peptide to the subjects, wherein some subjects areadministered a mast cell degranulation inhibitor to reduce aninflammatory response inducible by the internalization peptide and somesubjects are not depending on the dose of the pharmacological agentlinked to the internalization peptide with patients receiving a higherdose receiving the mast cell degranulation inhibitor.

The invention further provides a method of treating or effectingprophylaxis of a disease mediated by excitotoxicity comprisingadministering to a human subject having or at risk of the disease apeptide having an amino acid sequence of SEQ ID NO:6(YGRKKRRQRRRKLSSIESDV) at a dose of greater or equal to 2.0 mg/kg; andadministering to the subject a mast cell degranulation inhibitor,whereby the mast cell degranulation inhibitor can inhibit mast celldegranulation inducible by the internalization peptide and/or the mastcell degranulation inhibitor is administered within a period of 30minutes before to 15 minutes after the pharmacological agent.Optionally, the dose is 2.6 mg/kg.

The invention further provides a method of delivering a pharmacologicalagent linked to an internalization peptide to a subject having or a riskof a disease treatable by the pharmacological agent, comprisingadministering the pharmacological agent linked to the internalizationpeptide to the subject, administering a mast cell degranulationinhibitor and an anti-histamine to reduce an inflammatory responseinducible by the internalization peptide.

The invention provides methods of delivering a pharmacologic agent to asubject. The methods involve administering the pharmacologic agentlinked to an internalization peptide to the subject; and administering amast cell degranulation inhibitor to the subject, whereby the mast celldegranulation inhibitor can inhibit a mast cell degranulation inducibleby the internalization peptide and/or the mast cell degranulationinhibitor is administered within a period of 30 minutes before to 15minutes after the pharmacological agent. In some methods, the mast celldegranulation inhibitor inhibits a decline in blood pressure or skinrash induced by the internalization peptide. In some methods, the mastcell degranulation inhibitor is cromolyn. In some methods, the mast celldegranulation inhibitor is administered by a peripheral route. In somemethods, the mast cell degranulation inhibitor is administered within awindow of 30 minutes before to 15 minutes after administering thepharmacologic agent. In some methods, the mast cell degranulationinhibitor is administered within a window of 15 minutes before to thesame time as administering the pharmacologic agent. In some methods, thesubject is suffering from an episode of a disease and thepharmacological agent and the mast cell degranulation inhibitor areadministered once during the disease episode. In some methods, theadministration of the mast cell degranulation inhibitor does not comportwith a recurring regime of administering the mast cell degranulationinhibitor to the patient without the pharmacologic agent. In somemethods, the mast cell degranulation inhibitor does not cross the bloodbrain barrier in sufficient amounts to exert a detectablepharmacological effect in the brain when administered orally orintravenously. In some methods, the internalization peptide is a tatpeptide. In some methods, the tat peptide has an amino acid sequencecomprising RKKRRQRRR (SEQ ID NO:51), GRKKRRQRRR (SEQ ID NO:1),YGRKKRRQRRR (SEQ ID NO:2), FGRKKRRQRRR (SEQ ID NO:3) or GRKKRRQRRRP (SEQID NO:72). In some methods, the pharmacologic agent is a peptide,optionally, KLSSIESDV (SEQ ID NO:5).

The invention further provides a mast cell degranulation inhibitor foruse in inhibiting mast cell degranulation inducible inducible by aninternalization peptide linked to a pharmacological agent and/or for usein inhibiting a reduction in blood pressure inducible inducible by theinternalization peptide, and/or for use in inhibiting a skin rashinducible by the internalization peptide. Optionally, the mast celldegranulation inhibitor is administered within a period of 15 minutesbefore administrating the pharmacological agent or the mast celldegranulation inhibitor and pharmacological agent are administered byintravenous infusion at the same time. Optionally, the mast celldegranulation inhibitor is administered nasally. Optionally, wherein thedose of the pharmacological agent linked to the internalization peptideis greater than 2.6 mg/kg, optionally greater than 3 mg/kg or 5 mg/kg.Optionally, the mast cell degranulation inhibitor and pharmacologicalagent are administered once per episode of disease. In some uses, thedisease is characterized by cerebral ischemia. Optionally, the mast celldegranulation inhibitor is cromolyn and the pharmacological agent hasthe amino acid sequence YGRKKRRQRRRKLSSIESDV (SEQ ID NO:6).

The invention further provides a kit comprising a pharmacological agentlinked to an internalization peptide, and a mast cell degranulationinhibitor.

The invention further provides methods of treating or effectingprophylaxis of a disease mediated by excitotoxicity. The method involvesadministering to a subject having or at risk of the disease apharmacologic agent that inhibits binding of PSD95 to NDMAR 2B linked toan internalization peptide in a regime effective to treat or effectprophylaxis of the disease; and administering to the subject a mast celldegranulation inhibitor, whereby the mast cell degranulation inhibitorcan inhibit mast cell degranulation inducible by the internalizationpeptide and/or the mast cell degranulation inhibitor is administeredwithin a period of 30 minutes before to 15 minutes after thepharmacological agent. In some methods, the mast cell degranulationinhibitor inhibits a decline in blood pressure induced by theinternalization peptide. In some methods, the mast cell degranulationinhibits a decline in blood pressure induced by internalization peptide.Optionally, the pharmacological agent is a PL peptide of an NMDARreceptor. Optionally, the internalization peptide is a tat peptide.Optionally, the internalization peptide has an amino acid sequencecomprising RKKRRQRRR (SEQ ID NO:51), GRKKRRQRRR (SEQ ID NO:1),YGRKKRRQRRR (SEQ ID NO:2), FGRKKRRQRRR (SEQ ID NO:3) or GRKKRRQRRRPQ(SEQ ID NO:4). In some methods, the disease is stroke. In some methods,the subject is at risk of transient cerebral ischemic attack as a resultof undergoing surgery. In some methods, the mast cell degranulationinhibitor is cromolyn. In some methods, the mast cell degranulationinhibitor is administered by a peripheral route. In some methods, themast cell degranulation inhibitor is administered within a window of 30minutes before to 15 after administering the pharmacologic agent. Insome methods, the mast cell degranulation inhibitor is administeredwithin a window of 15 minutes before to the same time as administeringthe pharmacologic agent. In some methods, the subject is suffering froman episode of a disease and the pharmacological agent and the mast celldegranulation inhibitor are administered once during the diseaseepisode. In some methods, the administration of the mast celldegranulation inhibitor does not comport with a recurring regime ofadministering the mast cell degranulation inhibitor to the patientwithout the pharmacologic agent. In some methods, the mast celldegranulation inhibitor does not cross the blood brain barrier insufficient amounts to exert a pharmacological effect in the brain whenadministered orally or intravenously. In some methods, the mast celldegranulation inhibitor is administered nasally, orally orintravenously.

The invention also provides in a method of delivering a pharmacologicagent linked to an internalization peptide to a subject, the improvementwherein the internalization peptide is administered with a mast celldegranulation inhibitor that can inhibit mast cell degranulationinducible by the internalization peptide and/or the mast celldegranulation inhibitor is administered within a period of 30 minutesbefore to 15 minutes after the pharmacological agent. Optionally, theinternalization peptide is a tat peptide.

The invention further provides a method of inhibiting mast celldegranulation. The method involves administering a mast celldegranulation inhibitor to a subject who has been or will beadministered a pharmacologic agent linked to an internalization peptide;whereby the anti-inflammatory agent can inhibit mast cell degranulationinducible by the internalization peptide and/or the mast celldegranulation inhibitor is administered within a period of 30 minutesbefore to 15 minutes after the pharmacological agent. Optionally, themast cell degranulation inhibitor inhibits a decline in blood pressureinduced by the internalization peptide. Optionally, the mast celldegranulation inhibitor inhibits development of a skin rash induced bythe internalization peptide.

The invention further provides methods of delivering a pharmacologicagent to a subject. The method involves administering the pharmacologicagent linked to an internalization peptide to a subject, wherein thesubject has been or will be administered a mast cell degranulationinhibitor, whereby the mast cell degranulation inhibitor inhibits mastcell degranulation induced by the internalization peptide and/or themast cell degranulation inhibitor is administered within a period of 30minutes before to 15 minutes after the pharmacological agent. In somemethods, the mast cell degranulation inhibitor inhibits a decline inblood pressure induced by the internalization peptide.

The invention further provides methods of inhibiting inflammationinducible by a pharmacological agent linked to an internalizationpeptide, comprising administering a mast cell degranulation inhibitor atthe same time as or up to 15 minutes before the pharmacological agent.In some methods, the pharmacological agent and mast cell degranulationinhibitor are administered at the same time by intravenous infusion. Insome methods, the mast cell degranulation inhibitor is administeredbefore the pharmacological agent.

The invention provides a method of delivering a pharmacologic agent to asubject. The method comprises administering the pharmacologic agentlinked to an internalization peptide to the subject; and administeringan anti-inflammatory agent to the subject, whereby the anti-inflammatoryagent inhibits an inflammatory response induced by the internalizationpeptide. Optionally, the anti-inflammatory agent is an anti-histamine ora corticosteroid. Optionally, the internalization peptide is a tatpeptide. Optionally, the tat peptide has an amino acid sequencecomprising GRKKRRQRRR (SEQ ID NO:1), YGRKKRRQRRR (SEQ ID NO:2),FGRKKRRQRRR (SEQ ID NO:3), or GRKKRRQRRRPQ (SEQ ID NO:4). Optionally,the pharmacologic agent is a peptide. Optionally, the pharmacologicagent is KLSSIESDV (SEQ ID NO:5).

The invention also provides for use of an anti-inflammatory agent in themanufacture of a medicament to inhibit an inflammatory response inducedby an internalization peptide linked to a pharmacological agent.

The invention also provides a kit comprising a pharmacological agentlinked to an internalization peptide, and an anti-inflammatory agentthat inhibits an inflammatory response induced by the internalizationpeptide.

The invention also provides an internalization peptide linked to biotinhaving reduced capacity to induce an inflammatory response compared tothe internalization peptide without the biotin.

The invention also provides a method of delivering a pharmacologic agentto a subject, the method comprising administering the pharmacologicagent linked to an internalization peptide to the subject; wherein theinternalization peptide is biotinylated, and the biotinylation reducesthe capacity of the internalization peptide to induce an inflammatoryresponse relative to the internalization peptide without the biotin.

The invention also provides a method of treating or effectingprophylaxis of a disease mediated by excitotoxicity comprisingadministering to a subject having or at risk of the disease apharmacologic agent that inhibits binding of PSD95 to NDMAR 2B linked toan internalization peptide in a regime effective to treat or effectprophylaxis of the disease; and administering to the subject ananti-inflammatory agent, whereby the anti-inflammatory agent inhibits aninflammatory response induced by the internalization peptide.Optionally, the pharmacological agent is a PL peptide of an NMDARreceptor. Optionally, the internalization peptide is a tat peptide.Optionally, the internalization peptide has an amino acid sequencecomprising GRKKRRQRRR (SEQ ID NO:1), YGRKKRRQRRR (SEQ ID NO:2),FGRKKRRQRRR (SEQ ID NO:3) or GRKKRRQRRRPQ (SEQ ID NO:4). Optionally, thesubject is female. Optionally, the disease is stroke. In some methods,the subject is at risk of transient cerebral ischemic attack as a resultof undergoing heart surgery.

The invention further provides a method of treating or effectingprophylaxis of a disease mediated by excitotoxicity comprisingadministering to a subject having or at risk of the disease apharmacologic agent that inhibits binding of PSD95 to NDMAR 2B linked toan internalization peptide in a regime effective to treat or effectprophylaxis of the disease; wherein the internalization peptide isbiotinylated, and the biotinylation reduces the capacity of theinternalization peptide to induce an inflammatory response.

The invention further provides a method of treating or effectingprophylaxis of a disease mediated by excitotoxicity comprisingadministering to a female subject having or at risk of the disease apharmacologic agent that inhibits binding of PSD95 to NDMAR 2B linked toan internalization peptide in a regime effective to treat or effectprophylaxis of the disease. Optionally, the internalization peptide is atat peptide.

The invention further provides an improvement in a method of deliveringa pharmacologic agent linked to an internalization peptide to a subject,wherein either the internalization peptide is biotinylated oradministered with an immunosuppressive that inhibits an inflammatoryresponse induced by the internalization peptide. Optionally, theinternalization peptide is a tat peptide.

The invention further provides a method of inhibiting an inflammatoryresponse, the method comprising: administering an anti-inflammatoryagent to a subject who has been or will be administered a pharmacologicagent linked to an internalization peptide; whereby theanti-inflammatory agent inhibits an inflammatory response induced by theinternalization peptide.

The invention further provides a method of delivering a pharmacologicagent to a subject, the method comprising: administering thepharmacologic agent linked to an internalization peptide to a subject,wherein the subject has been or will be administered ananti-inflammatory agent, whereby the anti-inflammatory agent inhibits aninflammatory response induced by the internalization peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Gender difference in infarct size in the P3VO model of stroke inthe rat. Saline males: male stroke rats treated with saline (control).Tat-NR2B9c males: Male stroke rats treated with Tat-NR2B9c, i.e., thepeptide YGRKKRRQRRRKLSSIESDV (SEQ ID NO:6), containing both Tat sequenceand the 9 carboxy-terminal amino acids of the NR2B subunit. Salinefemales: female stroke rats treated with saline (control). Tat-NR2B9cfemales: Female stroke rats treated with Tat-NR2B9c, i.e., the peptideYGRKKRRQRRRKLSSIESDV (SEQ ID NO:6), containing both Tat sequence and 9carboxy-terminal amino acids of the NR2B subunit. Y axis: Size ofinfarct, measured (in percentage terms) relative to size of infarct inmale rats treated with saline alone)

FIG. 2: Peptides containing Tat sequence cause mast cell degranulation.CI: Calcium Ionophore (positive control). NA-1: Tat-NR2B9c, i.e., thepeptide YGRKKRRQRRRKLSSIESDV (SEQ ID NO:6), containing both Tat sequenceand the 9 carboxy-terminal amino acids of the NR2B subunit. NR2B9c:peptide KLSSIESDV (SEQ ID NO:5), PSD-95 binding sequence of the NMDANR2B subunit, devoid of the Tat sequence. AA: peptideYGRKKRRQRRRKLSSIEADA (SEQ ID NO:7), identical to Tat-NR2B9c, but with 2point mutations in the PSD-95 binding domain making it incapable ofbinding PSD-95. Degranulation was measured by relative tryptase activity(% over control). Bars indicate the means±S.D. of 3-6 independentreplicates.

FIG. 3: Mast cell degranulation by peptides containing Tat sequence isdose-dependent. CI: Calcium Ionophore (positive control). NA-1:Tat-NR2B9c, i.e., the peptide YGRKKRRQRRRKLSSIESDV (SEQ ID NO:6),containing both Tat sequence and the 9 carboxy-terminal amino acids ofthe NR2B subunit. AA: peptide YGRKKRRQRRRKLSSTEADA (SEQ ID NO:7),identical to Tat-NR2B9c, but with 2 point mutations in the PSD-95binding domain making it incapable of binding PSD-95.

FIG. 4: Mast cell degranulation by peptides containing Tat sequencevariants. CI: Calcium Ionophore (positive control). Tat-NR2B9c: thepeptide YGRKKRRQRRRKLSSIESDV (SEQ ID NO:6), containing both Tat sequenceand the 9 carboxy-terminal amino acids of the NR2B subunit. TAT: Tatpeptide sequence YGRKKRRQRRR (SEQ ID NO:2). 2B9c: peptide KLSSIESDV (SEQID NO:5), PSD-95 binding sequence of the NMDA NR2B subunit devoid of theTat sequence. AA: peptide YGRKKRRQRRRKLSSIEADA (SEQ ID NO:7), identicalto Tat-NR2B9c, but with 2 point mutations in the PSD-95 binding domainmaking it incapable of binding PSD-95. F-Tat-NR2B9c: peptideFGRKKRRQRRRKLSSIESDV (SEQ ID NO:8). Tat-NR2B9c K>A: YGRKKRRQRRRALSSIESDV(SEQ ID NO:9). F-Tat-NR2B9c K>A: FGRKKRRQRRRALSSIESDV (SEQ ID NO:10).

FIG. 5: Conjugates of peptides comprising Tat sequence fail to elicitmast cell degranulation.

FIG. 6: Observed drop in blood pressure observed after administration of50 mg/kg Tat-NR2B9c to beagle dogs.

FIG. 7 shows reduction in blood pressure after administration ofTat-NR2B9c or Rv-Tat-NR2B9c to rats.

FIGS. 8A-D show changes mean arterial pressure (MAP) followingadministration of cromolyn and Tat-NR2B9c. FIG. 8A shows a time course,FIG. 8B shows percentage change in area under the curve (AUC), FIG. 8Cshows MAP values after treatment with peptide transduction domain in thepresence and absence of Cromolyn, FIG. 8D shows the percentage of MAPtrough after treatment relative to MAP before treatment. FIGS. 8E (timecourse) and 8F (bar chart) show cromolyn has a similar effect withRv-Tat-NR2B9c.

FIGS. 9A-D provide similar data to FIGS. 8A-D except cromolyn isreplaced with dephenhydramine.

FIGS. 10A-D provide similar data to FIGS. 8A-D except cromolyn isreplaced by pyrilamine.

FIGS. 11A-D provide similar data to FIGS. 8A-D except cromolyn isreplaced with a combination of diphenhydramine and Ranitidine.

FIG. 12 is a schematic showing a cationic peptide, such as tat, inducingmast cell degranulation and consequent release of histamine and otherfactors, which cause diverse effects including a lowering of bloodpressure. Mast cell granulation inhibitors (also known as mast cellstabilizers) inhibit the degranulation of mast cells and consequentrelease of histamine and other molecules by the mast cells.

FIGS. 13A and 13B show a lodoxamide co-formulation with Tat-NR2B9c andcromolyn administered immediately before Tat-NR2B9c completely abrogatea drop in MAP due to Tat-NR2B9c.

FIG. 14 show that Rv-NR2B9c is effective in reducing infarcts in ananimal model of cerebral ischemia.

FIG. 15 shows that Tat-NR2B9c in combination with lodoxamide resulted ina statistically significant reduction relative to Tat-NR2B9c alone.

DETAILED DESCRIPTION Definitions

A “chimeric peptide” means a peptide having two component peptides notnaturally associated with one another joined to one another as a fusionprotein or by chemical linkage.

A “fusion” protein or polypeptide refers to a composite polypeptide,i.e., a single contiguous amino acid sequence, made up of sequences fromtwo (or more) distinct, heterologous polypeptides which are not normallyfused together in a single polypeptide sequence.

The term “PDZ domain” refers to a modular protein domain of about 90amino acids, characterized by significant sequence identity (e.g., atleast 60%) to the brain synaptic protein PSD-95, the Drosophila septatejunction protein Discs-Large (DLG), and the epithelial tight junctionprotein ZO1 (ZO1). PDZ domains are also known as Discs-Large homologyrepeats (“DHRs”) and GLGF repeats. PDZ domains generally appear tomaintain a core consensus sequence (Doyle, D. A., 1996, Cell 85:1067-76). Exemplary PDZ domain-containing proteins and PDZ domainsequences disclosed in U.S. application Ser. No. 10/714,537, which isherein incorporated by reference in its entirety.

The term “PL protein” or “PDZ Ligand protein” refers to a naturallyoccurring protein that forms a molecular complex with a PDZ-domain, orto a protein whose carboxy-terminus, when expressed separately from thefull length protein (e.g., as a peptide fragment of 3-25 residues, e.g.3, 4, 5, 8, 9, 10, 12, 14 or 16 residues), forms such a molecularcomplex. The molecular complex can be observed in vitro using the “Aassay” or “G assay” described, e.g., in U.S. application Ser. No.10/714,537, or in vivo.

The term “NMDA receptor,” or “NMDAR,” refers to a membrane associatedprotein that is known to interact with NMDA. The term thus includes thevarious subunit forms described herein. Such receptors can be human ornon-human (e.g., mouse, rat, rabbit, monkey).

A “PL motif” refers to the amino acid sequence of the C-terminus of a PLprotein (e.g., the C-terminal 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or25 contiguous residues) (“C-terminal PL sequence”) or to an internalsequence known to bind a PDZ domain (“internal PL sequence”).

A “PL peptide” is a peptide of comprising or consisting of, or otherwisebased on, a PL motif that specifically binds to a PDZ domain.

The terms “isolated” or “purified” means that the object species (e.g.,a peptide) has been purified from contaminants that are present in asample, such as a sample obtained from natural sources that contain theobject species. If an object species is isolated or purified it is thepredominant macromolecular (e.g., polypeptide) species present in asample (i.e., on a molar basis it is more abundant than any otherindividual species in the composition), and preferably the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, an isolated, purified orsubstantially pure composition comprises more than 8 to 90 percent ofall macromolecular species present in a composition. Most preferably,the object species is purified to essential homogeneity (i.e.,contaminant species cannot be detected in the composition byconventional detection methods), wherein the composition consistsessentially of a single macromolecular species. The term isolated orpurified does not necessarily exclude the presence of other componentsintended to act in combination with an isolated species. For example, aninternalization peptide can be described as isolated notwithstandingthat it is linked to an active peptide.

A “peptidomimetic” refers to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics of apeptide consisting of natural amino acids. The peptidomimetic cancontain entirely synthetic, non-natural analogues of amino acids, or canbe a chimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The peptidomimetic can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetic's structure and/or inhibitory or binding activity. Polypeptidemimetic compositions can contain any combination of nonnaturalstructural components, which are typically from three structural groups:a) residue linkage groups other than the natural amide bond (“peptidebond”) linkages; b) non-natural residues in place of naturally occurringamino acid residues; or c) residues which induce secondary structuralmimicry, i.e., to induce or stabilize a secondary structure, e.g., abeta turn, gamma turn, beta sheet, alpha helix conformation, and thelike. In a peptidomimetic of a chimeric peptide comprising an activepeptide and an internalization peptide, either the active moiety or theinternalization moiety or both can be a peptidomimetic.

Individual peptidomimetic residues can be joined by peptide bonds, otherchemical bonds or coupling means, such as, e.g., glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,N,N-dicyclohexylcarbodiimide (DCC) or N,N-diisopropylcarbodiimide (DIC).Linking groups that can be an alternative to the traditional amide bond(“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN⁴⁻-), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, A Peptide Backbone Modifications, Marcell Dekker, N.Y.).

Mimetics of aromatic amino acids can be generated by replacing by, e.g.,D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine;D- or L-1, -2,3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluorophenylalanine; D- or L-p-biphenylphenylalanine; K- orL-p-methoxybiphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anonnatural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R—N—C—N—R═) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions.

Mimetics of basic amino acids can be generated by substitution with,e.g., (in addition to lysine and arginine) the amino acids ornithine,citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid,where alkyl is defined above. Nitrile derivative (e.g., containing theCN-moiety in place of COOH) can be substituted for asparagine orglutamine. Asparaginyl and glutaminyl residues can be deaminated to thecorresponding aspartyl or glutamyl residues.

Arginine residue mimetics can be generated by reacting arginyl with,e.g., one or more conventional reagents, including, e.g., phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably underalkaline conditions.

Tyrosine residue mimetics can be generated by reacting tyrosyl with,e.g., aromatic diazonium compounds or tetranitromethane.N-acetylimidizol and tetranitromethane can be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.

Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole.

Lysine mimetics can be generated (and amino terminal residues can bealtered) by reacting lysinyl with, e.g., succinic or other carboxylicacid anhydrides. Lysine and other alpha-amino-containing residuemimetics can also be generated by reaction with imidoesters, such asmethyl picolinimidate, pyridoxal phosphate, pyridoxal,chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4,pentanedione, and transamidase-catalyzed reactions with glyoxylate.

Mimetics of methionine can be generated by reaction with, e.g.,methionine sulfoxide. Mimetics of proline include, e.g., pipecolic acid,thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline,3- or 4-methylproline, or 3,3,-dimethylproline. Histidine residuemimetics can be generated by reacting histidyl with, e.g.,diethylprocarbonate or para-bromophenacyl bromide.

Other mimetics include, e.g., those generated by hydroxylation ofproline and lysine; phosphorylation of the hydroxyl groups of seryl orthreonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

The mimetics of the invention can also include compositions that containa structural mimetic residue, particularly a residue that induces ormimics secondary structures, such as a beta turn, beta sheet, alphahelix structures, gamma turns, and the like. For example, substitutionof natural amino acid residues with D-amino acids; N-alpha-methyl aminoacids; C-alpha-methyl amino acids; or dehydroamino acids within apeptide can induce or stabilize beta turns, gamma turns, beta sheets oralpha helix conformations. Beta turn mimetic structures have beendescribed, e.g., by Nagai (1985) Tet. Lett. 26:647-650; Feigl (1986) J.Amer. Chem. Soc. 108:181-182; Kahn (1988) J. Amer. Chem. Soc.110:1638-1639; Kemp (1988) Tet. Lett. 29:5057-5060; Kahn (1988) J.Molec. Recognition 1:75-79. Beta sheet mimetic structures have beendescribed, e.g., by Smith (1992) J. Amer. Chem. Soc. 114:10672-10674.For example, a type VI beta turn induced by a cis amide surrogate,1,5-disubstituted tetrazol, is described by Beusen (1995) Biopolymers36:181-200. Incorporation of achiral omega-amino acid residues togenerate polymethylene units as a substitution for amide bonds isdescribed by Banerjee (1996) Biopolymers 39:769-777. Secondarystructures of polypeptides can be analyzed by, e.g., high-field .sup.1HNMR or 2D NMR spectroscopy, see, e.g., Higgins (1997) J. Pept. Res.50:421-435. See also, Hruby (1997) Biopolymers 43:219-266, Balaji, etal., U.S. Pat. No. 5,612,895.

The term “specific binding” refers to binding between two molecules, forexample, a ligand and a receptor, characterized by the ability of amolecule (ligand) to associate with another specific molecule (receptor)even in the presence of many other diverse molecules, i.e., to showpreferential binding of one molecule for another in a heterogeneousmixture of molecules. Specific binding of a ligand to a receptor is alsoevidenced by reduced binding of a detectably labeled ligand to thereceptor in the presence of excess unlabeled ligand (i.e., a bindingcompetition assay).

Excitotoxicity is the pathological process by which neurons are damagedand killed by the overactivation of receptors for the excitatoryneurotransmitter glutamate, such as the NMDA receptors, for instanceNMDAR 2B.

The term “subject” includes humans and veterinary animals, such asmammals.

The term “agent” includes any element, compound, or entity, including,e.g., pharmaceutical, therapeutic, pharmacologic, cosmeceutical, drug,toxin, natural product, synthetic compound, chemical compounds. Agentscan be biologics (e.g., peptides, petidomimetics, or antibodies) ororganic small molecules (usually less than 500 Da) among others.

The term “pharmacologic agent” means an agent having a pharmacologicalactivity. Agents include compounds that are known (i.e., approved by FDAor similar body in other countries) drugs, compounds for whichpharmacological activity has been identified but which are undergoingfurther therapeutic evaluation. A chimeric agent comprises apharmacologic agent linked to an internalization peptide. An agent canbe described as having pharmacological activity if it exhibits anactivity in a screening system that indicates that the active agent isor may be useful in the prophylaxis or treatment of a disease. Thescreening system can be in vitro, cellular, animal or human. Agents canbe described as having pharmacological activity notwithstanding thatfurther testing may be required to establish actual prophylactic ortherapeutic utility in treatment of a disease.

A tat peptide means a peptide comprising or consisting of GRKKRRQRRR(SEQ ID NO:1), in which no more than 5 residues are deleted, substitutedor inserted within the sequence, which retains the capacity tofacilitate uptake of a linked peptide or other agent into cells.Preferably any amino acid changes are conservative substitutions.Preferably, any substitutions, deletions or internal insertions in theaggregate leave the peptide with a net cationic charge, preferablysimilar to that of the above sequence. The amino acids of a tat peptidecan be derivatized with biotin or similar molecule to reduce aninflammatory response, as described further below.

Co-administration of a pharmacological agents linked to aninternalization peptide and an anti-inflammatory agent means that thetwo agents are administered sufficiently proximately in time that theanti-inflammatory agent can inhibit an inflammatory response inducibleby the internationalization peptide.

Statistically significant refers to a p-value that is <0.05, preferably<0.01 and most preferably <0.001.

I. General

The invention provides methods of delivering pharmacologic agents linkedto an internalization peptide, in which an inflammatory responseinducible by the internalization peptide is inhibited byco-administration of an anti-inflammatory or by linking theinternalization peptide to biotin or similar molecule. Such methods arepremised in part on the results described in the examples wherebyadministration of a pharmacological agent linked to tat at high dosagesis closely followed by an inflammatory response, which includes mastcell degranulation, histamine release and the typical sequelae ofhistamine release, such as redness, heat, swelling, and hypotension.Although practice of the methods of the invention is not dependent on anunderstanding of mechanism, it is believed that the mast celldegranulation is triggered by direct interaction between the cationictat peptide and mast cells rather than being triggered by an IgEantibody response. The inflammatory response can be inhibited byco-administering an anti-inflammatory agent, particularly a mast celldegranulation inhibitor, such as cromolyn, with the pharmacologicalagent linked to tat or other internalization peptide. Otheranti-inflammatory agents including anti-histamines and corticosteriodscan also be used. Alternatively, the inventors have found that thecapacity of internalization peptides to induce an inflammatory responsecan be reduced by linking them to biotin or similar molecule.

The invention further provides method of treating or effectingprophylaxis of diseases characterized by excitotoxicity, such as stroke.Such diseases can be treated using a pharmacologic agent that inhibitsinteraction between NMDARs with postsynaptic density 95 protein linkedto an internalization peptide. Preferably, in such methods, thepharmacologic agent is co-administered with an anti-inflammatory agent,preferably a mast cell granulation inhibitor, such as cromolyn, toinhibit an immune response inducible by the internalization peptide, orthe internalization peptide is linked to biotin or similar molecule, forthe reasons discussed above. Irrespective whether an anti-inflammatoryagent or biotinylated internalization peptide is used in such methods,the treatment or prophylaxis can be administered to both male and femalesubjects. The administration to female subjects is premised in part onresults described in the example in which the treatment in a rat modelof stroke was found to be at least as effective in female subjects asmale. The feasibility of administering a pharmacological agent thatinhibits interactions between PSD95 and NMDAR to a female subjectcontrasts with previous results in which inhibitors of nNOS were foundineffective to treat excitotoxic disease in female subjects.Administration of nNOS inhibitors were reported to protect againstdamaging effects of stroke in male rats, but increased cell injury infemale rats in an MCAO model. McCullough et al., Journal of CerebralBlood Flow & Metabolism, 25: 502-512 (2005).

II. Pharmacologic Agents

Internalization peptides can be linked to any pharmacologic agent topromote uptake of the agent through cell membranes, intracellularmembranes such as the nuclear membrane, and/or the blood brain barrier.The attachment of an internalization peptide to a pharmacologic agentimproves bioavailability at the intended site relative to use of thepharmacologic agent alone. The increased delivery due to the attachedinternalization peptides can allow decreased doses of pharmacologicagents, effective targeting of pharmacologic agents to a specific cellcompartment such as the nucleus, and/or reduced toxicity due to the useof lower doses.

Internalization peptides are particularly useful for pharmacologicagents that are required to enter cells and/or the nucleus.Pharmacologic agents that have poor bioavailabilty, high dosages orshort half-lives, or neuroactive drugs that need to cross the bloodbrain barrier to exert activity, are especially suitable for attachmentof internalization peptides. Peptides are one type of pharmacologicagent that are amenable to attachment of internalization, for instancethrough the use of a peptide bond that results in a chimeric peptidecomprising an amino acid sequence derived from the pharmacologic agent,and an amino acid sequence of the internalization peptide. Nucleicacids, and small organic molecules (less than 500 Da) are other examplesof compounds that can be linked to internalization peptides.

Some guidance for selection of pharmacologic agents, methods forattachments and use thereof is provided by the scientific and patentliterature relating to internalization peptides, such as tat (see, e.g.,U.S. Pat. No. 6,316,003 and U.S. Pat. No. 5,804,604). The table belowlists the names of pharmacologic agents (some of which are approveddrugs), the disorders they are useful for treating, whether the diseaseis acute or chronic, the routes of administration of drugs (to theextent established) and comments on problems with existing drugs thatmay in part be overcome by the improved transport through membranesconferred by an internalization peptide.

TABLE 1 Pharmacologic Acute/ Route of agent Disease chronic adminComment Reference Phenobarbitol Epilepsy IV/oral Dependence, Motamedi &(luminal sodium) tolerance Meador (2006) issues, Curr Neurolinteractions, Neurosci Rep, side effects, 6(4): 341-6. birth defectsDrugs.com Primidone Epilepsy Oral Side effects, Koristkova, et al(myidone, interactions (2006) Int J Clin mysoline) Pharmacol Ther,44(9): 438-42. Drugs.com Diazepam Anxiety IP/oral Dependence, Beard, etal (valium) side effects, (2003) Health interactions Technol Assess,7(40): iii, ix-x, 1- 111. Drugs.com Dopamine Parkinson's Cannot crossAhlskog (2001) BBB, side Neurol Clin, effects 19(3): 579-605. Drugs.comLevodopa Parkinson's Degraded Nyholm (2006) before BBB, Clin sideeffects, Pharmacokinet, halflife = 1.5 45(2): 109-36. hrs USPTO.gov(U.S. Pat. No. 7,160,913) Apomorphine IP Short half-life Nyholm (2006)Clin Pharmacokinet, 45(2): 109-36. Drugs.com Tirilazad mesylate StrokeIP Low efficacy, Hickenbottom & (Freedox) phase III Grotta (1998)stopped Semin Neurol 18(4): 485-92. Strokecenter.org Cyclosporine ImmuneIP Peptide, 5-18 Kees, et al (Gengraf) suppression hr halflife (2006)Ther Drug Monit, 28(3): 312-20. Drugs.com Vacomycin Antibiotic IPPeptide, low de Hoog, et al uptake, 4-6 hr (2004) Clin halflifePharmacokinet, 43(7): 417-40. Drugs.com Lisinopril Hyper- IP/oralPeptide, poor Tan, et al (2005) (Prinivil) tension BBB crossing, Am JHypertens, 12 hr halflife 18(2): 158-64. Drugs.com AzidothymidineAntiviral Oral Poor BBB Spitzenberger, et (AZT, zidoridine, crossing,05-3 al (2006) J Cereb combivir) hr halflife, Blood Flow hematologicMetab, Oct 25, toxicology Epub ahead of print. Drugs.com Piracetam Pain/Cannot cross Loscher & epilepsy BBB Potschka (2002) J Pharmacol ExpTher, 301(1): 7- 14. USPTO.gov (U.S. Pat. No. 7,157,421) NatrecorCardio-renal IV Unknown Feldman & Sun (BNP peptide) decom- efficacy(2004) Heart Fail pensation Rev, 9(3): 203-8. syndromeClinicaltrials.gov AVR-118 (peptide) Cancer Sub- UnknownClinicaltrials.gov palliative cutaneous efficacy, unknown dosageOxytocin (peptide) Mood IV/IM Interactions, Swaab, et al disordersunknown (2005) Ageing dosage Res Rev, 4(2): 141-94. Drugs.comPravastatin MS Oral Unknown Hatanaka (2000) (Pravachol) efficacy, lowClin bioavailability Pharmacokinet, 39(6): 397-412. Clinicaltrials.govRemifentanil Pain, burn IV 3.5 min Scott & Perry halflife, (2005) Drugs,metabolized by 65(13): 1793- unknown 1823. esterase Clinicaltrials.govNeurotensin Schizo- 13AA peptide, Boules, et al, phrenia, easily (2006)Peptides, Parkinson's, degraded, 27(10): 2523-33. addiction cannot crossBBB GDNF (glial Parkinson's Chronic Intra- Peptide, Grondin, et alderived parenchymal Cannot cross (2003) Prog neurotrophic BBB Drug Res,61: factor) 101-23. Protease inhibitors HIV Oral All HIV Oldfield &lopinavir protease Plosker (2006) ritonavir inhibitors Drugs 66(9):saquinavir suffer from the 1275-99. darunavir acute capacity Porter &atazanavir of HIV to Charman (2001) amprenavir mutate, Adv Drug Delivgenerating drug Rev, Oct 1; 50 resistant HIV Suppl 1: S127- strains 47.Piacenti (2006) Pharmacotherapy 26(8): 1111-33. DihydroergotamineMigraine IV, IM, sub- Interactions Modi & Lowder Q cause (2006) Am Famperipheral Physician 73(1): ischemia, 9 hr 72-8. halflife SporamaxAntifungal Oral Drug resistance Wang & Remold (itaconazole) eventually(2006) Cardiol develops, Rev 14(5): 223- congestive 6. heart failure insome populations Protein Kinase C Acute US pat inhibitors myocardialpublications infarction, 20050267030, stroke, 20060148702, ischemia,20060293237, reperfusion 20050215483, injury 20040204364, 20040009922AII-7 Cancer, Chronic Peptidomimetic Kunz et al, Mol Breast cancer thatblocks Cancer Res Erbb2 2006; 4(12): 983- intracellular 98 domain andincreases taxol sensitivity CRAMP peptide Salmonella IntracellularRosenberger, infection anti-microbial CM. PNAS | peptide that Feb. 24,reduces 2004 | vol. 101 | Salmonella no. 8 | 2422- replication 2427Sodium channel May reduce Peptide Vassilev, peptide muscle correspondingScience (1988) spasms to the short 241: 1658-6 (epilepsy, intracellularrestless leg, segment Parkinson's, between etc) homologous transmembranedomains III and IV of sodium channel alpha subunit slowed inactivationAptamer KDI1 Blocks EGF Buerger. J. Biol. signaling - Chem., Vol. 278,possible anti Issue 39, 37610- cancer 37621, Sep. 26, 2003 TransporterTurner et al RNA/gene therapy peptides can be (2007) Blood used to bringin Cells Mol Dis, RNAs or 38(1): 1-7. siRNA/RNAi for treatment

One class of agents of particular interest inhibits interactions betweenPSD-95 and one or more NMDARs. Such agents are useful for reducingdamaging effects of stroke and other neurological conditions mediated atleast in part by NMDAR excitotoxicity. Such agents include peptideshaving an amino acid sequence including or based on the PL motif of aNMDA Receptor or PDZ domain of PSD95. Such peptides can also oralternatively inhibit interactions between PSD-95 and nNOS and otherglutamate receptors (e.g., kainite receptors or AMPA receptors).Preferred peptides inhibit interaction between PDZ domains 1 and 2 ofpostsynaptic density-95 protein (PSD-95) (human amino acid sequenceprovided by Stathakism, Genomics 44(1):71-82 (1997)) and the C-terminalPL sequence of one or more NMDA Receptor 2 subunits including the NR2Bsubunit of the neuronal N-methyl-D-aspartate receptor (Mandich et al.,Genomics 22, 216-8 (1994)). NMDAR2B has GenBank ID 4099612, a C-terminal20 amino acids FNGSSNGHVYEKLSSIESDV (SEQ ID NO:11) and a PL motif ESDV(SEQ ID NO:12). Preferred peptides inhibit the human forms of PSD-95 andhuman NMDAR receptors. However, inhibition can also be shown fromspecies variants of the proteins. A list of NMDA and glutamate receptorsthat can be used appears below:

TABLE 2 NMDA Receptors With PL Sequences C-terminal C-terminalinternal PL Name GI or Acc# 20 mer sequence 4 mer sequence PL? ID NMDAR1307302 HPTDITGPLNLSDPSVST STVV X AA216 VV (SEQ ID NO: 13) (SEQ IDNO: 27) NMDAR1-1 292282 HPTDITGPLNLSDPSVST STVV X AA216VV (SEQ ID NO: 13) (SEQ ID NO: 27) NMDAR1-4 472845 HPTDITGPLNLSDPSVSTSTVV X AA216 VV (SEQ ID NO: 13) (SEQ ID NO: 27) NMDAR1- 2343286HPTDITGPLNLSDPSVST STVV X AA216 3b VV (SEQ ID NO: 13) (SEQ ID NO: 27)NMDAR1- 2343288 HPTDITGPLNLSDPSVST STVV X AA216 4b VV (SEQ ID NO: 13)(SEQ ID NO: 27) NMDAR1-2 11038634 RRAIHREEGQLQLCSRH HRESRES (SEQ ID NO: 14) (SEQ ID NO: 28) NMDAR1-3 11038636 RRAIHREEGQLQLCSRHHRES RES (SEQ ID NO: 14) (SEQ ID NO: 28) NMDAR2C 6006004TQGFPGPCTWRRISSLES ESEV X AA180 EV (SEQ ID NO: 15) (SEQ ID NO: 29)NMDAR3 560546 FNGSSNGHVYEKLSSIES ESDV X AA34.1 DV (SEQ ID NO: 11)(SEQ ID NO: 12) NMDAR3A 17530176 AVSRKTELEEYQRTSRT TCESCES (SEQ ID NO: 16) (SEQ ID NO: 30) NMDAR2B 4099612 FNGSSNGHVYEKLSSIESESDV X DV (SEQ ID NO: 11) (SEQ ID NO: 12) NMDAR2A 558748LNSCSNRRVYKKMPSIE ESDV X AA34.2 SDV (SEQ ID NO: 17) (SEQ ID NO: 12)NMDAR2D 4504130 GGDLGTRRGSAHFSSLE ESEV X SEV (SEQ ID NO: 18) (SEQ IDNO: 29) Glutamate AF009014 QPTPTLGLNLGNDPDRG GTSI (SEQ ID Xreceptor delta TSI (SEQ ID NO: 19) NO: 31) 2 Glutamate 128953MQSIPCMSHSSGMPLGA ATGL (SEQ X receptor 1 TGL (SEQ ID NO: 20) ID NO: 32)Glutamate L20814 QNFATYKEGYNVYGIES SVKI (SEQ ID X receptor 2VKI (SEQ ID NO: 21) NO: 33) Glutamate AF167332 QNYATYREGYNVYGTESVKI (SEQ ID X receptor 3 SVKI (SEQ ID NO: 22) NO: 33) Glutamate U16129HTGTAIRQSSGLAVIASD SDLP (SEQ ID receptor 4 LP (SEQ ID NO: 23) NO: 34)Glutamate U16125 SFTSILTCHQRRTQRKET ETVA (SEQ X receptor 5VA (SEQ ID NO: 24) ID NO: 35) Glutamate U16126 EVINMHTFNDRRLPGKEETMA (SEQ X receptor 6 TMA (SEQ ID NO: 25) ID NO: 36) Glutamate U16127RRLPGKDSMACSTSLAP PVFP (SEQ ID receptor 7 VFP (SEQ ID NO: 26) NO: 37)

Some peptides inhibit interactions between PSD-95 and multiple NMDARsubunits. In such instances, use of the peptide does not necessarilyrequire an understanding of the respective contributions of thedifferent NMDARs to excitatory neurotransmission. Other peptides arespecific for a single NMDAR. Similarly, if an agent characterized asinhibiting one interaction (e.g., PSD-95 and NMDAR) inherently inhibitsanother interaction (e.g., PSD-95 and nNOS), uses or methods employingthe agent can be effected by a mechanism that involves either or bothinhibitions.

Peptides can include or be based on a PL motif from the C-terminus ofany of the above subunits and have an amino acid sequence comprising[S/T]-X-[V/L]. This sequence preferably occurs at the C-terminus of thepeptides of the invention. Preferred peptides have an amino acidsequence comprising [E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L] (SEQ ID NO:38) attheir C-terminus. Exemplary peptides comprise: ESDV (SEQ ID NO:12), ESEV(SEQ ID NO:29), ETDV (SEQ ID NO:39), ETEV (SEQ ID NO:40), DTDV (SEQ IDNO:41), and DTEV (SEQ ID NO:42) as the C-terminal amino acids. Twoparticularly preferred peptides are KLSSIESDV (SEQ ID NO:5), andKLSSIETDV (SEQ ID NO:43). Such peptides usually have 3-25 amino acids(without an internalization peptide), peptide lengths of 5-10 aminoacids, and particularly 9 amino acids (also without an internalizationpeptide) are preferred. In some such peptides, all amino acids are fromthe C-terminus of an NMDA receptor (not including amino acids from aninternalization peptide).

Other peptides that inhibit interactions between PDS95 and NDMARsinclude peptides from PDZ domain 1 and/or 2 of PSD-95 or a subfragmentof any of these that inhibits interactions between PSD-95 and an NMDAreceptor, such as NMDA 2B. Such active peptides comprise at least 50,60, 70, 80 or 90 amino acids from PDZ domain 1 and/or PDZ domain 2 ofPSD-95, which occur within approximately amino acids 65-248 of PSD-95provided by Stathakism, Genomics 44(1):71-82 (1997) (human sequence) orNP_(—)031890.1, GI:6681195 (mouse sequence) or corresponding regions ofother species variants.

Peptides and peptidomimetics of the invention can contain modified aminoacid residues for example, residues that are N-alkylated. N-terminalalkyl modifications can include e.g., N-Methyl, N-Ethyl, N-Propyl,N-Butyl, N-Cyclohexylmethyl, N-Cyclyhexylethyl, N-Benzyl, N-Phenylethyl,N-phenylpropyl, N-(3,4-Dichlorophenyl)propyl,N-(3,4-Difluorophenyl)propyl, and N-(Naphthalene-2-yl)ethyl).

Bach, J. Med. Chem. 51, 6450-6459 (2008) and WO 2010/004003 hasdescribed a series of analogs of NR2B9c. PDZ-binding activity isexhibited by peptides having only three C-terminal amino acids (SDV).Bach also reports analogs having an amino acid sequence comprising orconsisting of YtSXV (SEQ ID NO:68), wherein t and S are alternativeamino acids, Y is selected from among E, Q, and A, or an analoguethereof, X is selected from among A, Q, D, N,N-Me-A, N-Me-Q, N-Me-D, andN-Me-N or an analogue thereof. Optionally the peptide is N-alkylated inposition P3 position (third amino acid from C-terminus, i.e., the tSposition). The peptide can be N-alkylated with a cyclohexane or aromaticsubstituent, and further comprises a spacer group between thesubstituent and the terminal amino group of the peptide or peptideanalogue, wherein the spacer is an alkyl group, preferably selected fromamong methylene, ethylene, propylene and butylene. The aromaticsubstituent can be a naphthalen-2-yl moiety or an aromatic ringsubstituted with one or two halogen and/or alkyl group.

Other modifications can also be incorporated without adversely affectingthe activity and these include substitution of one or more of the aminoacids in the natural L-isomeric form with amino acids in the D-isomericform. Thus, any amino acid naturally occurring in the L-configuration(which can also be referred to as the R or S, depending upon thestructure of the chemical entity) can be replaced with the amino acid ofthe same chemical structural type or a peptidomimetic, but of theopposite chirality, generally referred to as the D-amino acid, but whichcan additionally be referred to as the R- or S-form. Thus, apeptidomimetic may include 1, 2, 3, 4, 5, at least 50%, or all D-aminoacid resides. A peptidomimetic containing some or all D residues issometimes referred to an “inverso” peptide.

Peptidomimetics also include retro peptides. A retro peptide has areverse amino acid sequence. Peptidomimetics also include retro inversopeptides in which the order of amino acids is reversed from so theoriginally C-terminal amino acid appears at the N-terminus and D-aminoacids are used in place of L-amino acids. WO 2008/014917 describes aretro-inverso analog of Tat-NR2B9c having the amino acid sequencevdseisslk-rrrqukkrgyin (SEQ ID NO: 69) (lower case letters indicating Damino acids), and reports it to be effective in inhibiting cerebralischemia. Another effective peptide described herein is Rv-Tat-NR2B9c(RRRQRRKKRGYKLSSIESDV SEQ ID NO:70).

A linker, e.g., a polyethylene glycol linker, can be used to dimerizethe active moiety of the peptide or the peptidomimetic to enhance itsaffinity and selectivity towards proteins containing tandem PDZ domains.See e.g., Bach et al., (2009) Angew. Chem. Int. Ed. 48:9685-9689 and WO2010/004003. A PL motif-containing peptide is preferably dimerized viajoining the N-termini of two such molecules, leaving the C-termini free.Bach further reports that a pentamer peptide IESDV (SEQ ID NO:71) fromthe C-terminus of NMDAR 2B was effective in inhibiting binding of NMDAR2B to PSD95. Optionally, about 2-10 copies of a PEG can be joined intandem as a linker.

Appropriate pharmacological activity of peptides, peptidomimetics orother agent can be confirmed, if desired, using the animal modeldescribed in the Examples. Optionally, peptides or peptidomimetics canalso be screened for capacity to inhibit interactions between PSD-95 andNMDAR 2B using assays described in e.g., US 20050059597, which isincorporated by reference. Useful peptides typically have IC50 values ofless than 50 μM, 25 μM, 10 μM, 0.1 μM or 0.01 μM in such an assay.Preferred peptides typically have an 1050 value of between 0.001-1 μM,and more preferably 0.05-0.5 or 0.05 to 0.1 μM.

Peptides such as those just described can optionally be derivatized(e.g., acetylated, phosphorylated and/or glycosylated) to improve thebinding affinity of the inhibitor, to improve the ability of theinhibitor to be transported across a cell membrane or to improvestability. As a specific example, for inhibitors in which the thirdresidue from the C-terminus is S or T, this residue can bephosphorylated before use of the peptide.

Pharmacological agents also include small molecules that inhibitinteractions between PSD95 and NMDAR 2B, and/or other interactionsdescribed above. Suitable small-molecule inhibitors are described in WO07/079,406 and 60/947,892 filed on Jul. 3, 2007, each incorporated byreference in its entirety. These molecules were identified by in silicoscreening of a compound library for binding to PSD95, and binding ofexemplary compounds was verified experimentally.

Many appropriate compounds are described in U.S. Provisional App. No.60/947,883, hereby incorporated by reference in its entirety. Anexemplary class of suitable compounds are of the formula:

-   -   wherein R¹ is a member selected from the group consisting of        cyclohexyl substituted with 0-4 R⁷, phenyl substituted with 0-4        R⁷, —(CH₂)_(u)—(CHR⁸R⁹), a branched C₁₋₆ alkyl (isopropyl,        isobutyl, 1-isopropyl-2-methyl-butyl, 1-ethyl-propyl), and        —NH—C(O)—(CR¹⁰R¹¹)_(v)H;    -   each R⁷ is independently a member selected from the group        consisting of C₁₋₆ alkyl, C₁₋₆ alkoxy, —C(O)R¹², OH, COOH, —NO,        N-substituted indoline and a cell membrane translocation        peptide;    -   each R⁸ and R⁹ is independently selected from the group        consisting of H, OH, cyclohexane, cyclopentane, phenyl,        substituted phenyl and cyclopentadiene;    -   each R¹⁰ and R¹¹ is independently selected from the group        consisting of H, cyclohexane, phenyl and a cell membrane        translocation peptide;    -   R¹² is a member selected from the group consisting of C₁₋₆ alkyl        and aryl; and

each of u and v are independently from 0 to 20;

-   -   wherein one of R², R³, R⁴, R⁵ and R⁶ is —COOH, and wherein the        remainder of R², R³, R⁴, R⁵ and R⁶ are each independently        selected from the group consisting of F, H, OCH₃ and CH₃.

One such compound is 0620-0057, the structure of which is:

III. Internalization Peptides

Internalization peptides, also known as cell membrane transductionpeptides or cell penetrating peptides, are a well-known class ofrelatively short (e.g., 5-30 amino acids) peptides that allow manycellular or viral proteins to traverse membranes. Such peptidestypically have a cationic charge from an above normal representation(relative to proteins in general) of arginine and/or lysine residuesthat is believed to facilitate their passage across membranes. Some suchpeptides have at least 5, 6, 7 or 8 arginine and/or lysine residues.Examples include the antennapedia protein (Bonfanti, Cancer Res. 57,1442-6 (1997)) (and variants thereof), the tat protein of humanimmunodeficiency virus, the protein VP22, the product of the UL49 geneof herpes simplex virus type 1, Penetratin, SynB1 and 3, Transportan,Amphipathic, gp41NLS, polyArg, and several plant and bacterial proteintoxins, such as ricin, abrin, modeccin, diphtheria toxin, cholera toxin,anthrax toxin, heat labile toxins, and Pseudomonas aeruginosa exotoxin A(ETA). Other examples are described in the following references(Temsamani, Drug Discovery Today, 9(23):1012-1019, 2004; De Coupade,Biochem J., 390:407-418, 2005; Saalik Bioconjugate Chem. 15: 1246-1253,2004; Zhao, Medicinal Research Reviews 24(1):1-12, 2004; Deshayes,Cellular and Molecular Life Sciences 62:1839-49, 2005) (all incorporatedby reference).

A preferred internalization peptide is tat from the HIV virus. A tatpeptide reported in previous work comprises or consists of the standardamino acid sequence YGRKKRRQRRR (SEQ ID NO:2) found in HIV Tat protein.If additional residues flanking such a tat motif are present (beside thepharmacological agent) the residues can be for example natural aminoacids flanking this segment from a tat protein, spacer or linker aminoacids of a kind typically used to join two peptide domains, e.g., gly(ser)₄ (SEQ ID NO:44), TGEKP (SEQ ID NO:45), GGRRGGGS (SEQ ID NO:46), orLRQRDGERP (SEQ ID NO:47) (see, e.g., Tang et al. (1996), J. Biol. Chem.271, 15682-15686; Hennecke et al. (1998), Protein Eng. 11, 405-410)), orcan be any other amino acids that do not significantly reduce capacityto confer uptake of the variant without the flanking residues.Preferably, the number of flanking amino acids other than an activepeptide does not exceed ten on either side of YGRKKRRQRRR (SEQ ID NO:2).One suitable tat peptide comprising additional amino acid residuesflanking the C-terminus of YGRKKRRQRRR (SEQ ID NO:2) is YGRKKRRQRRRPQ(SEQ ID NO:48). However, preferably, no flanking amino acids arepresent.

Variants of the above tat peptide having reduced capacity to bind toN-type calcium channels are described by WO/2008/109010. Such variantscan comprise or consist of an amino acid sequence XGRKKRRQRRR (SEQ IDNO:49), in which X is an amino acid other than Y or nothing (in whichcase G is a free N-terminal residue). A preferred tat peptide has theN-terminal Y residue substituted with F. Thus, a tat peptide comprisingor consisting of FGRKKRRQRRR (SEQ ID NO:3) is preferred. Anotherpreferred variant tat peptide consists of GRKKRRQRRR (SEQ ID NO:1).Other tat peptides that can be used include GRKKRRQRRRPQ (SEQ ID NO:4)and GRKKRRQRRRP (SEQ ID NO:72). Other tat peptides comprises at leasteight contiguous amino acids of the sequence GRKKRRQRRR. Other tatpeptides that facilitate uptake of a pharmacological agent withoutinhibiting N-type calcium channels include those presented in Table 3.Another preferred tat peptide is referred to as rv-tat or RRRQRRKKRGY(amino acids 1-11 of SEQ ID NO:70).

TABLE 3 X-FGRKKRRQRRR (F-Tat) (SEQ ID NO: 3)X-GKKKKKQKKK (SEQ ID NO: 50) X-RKKRRQRRR (SEQ ID NO: 51)X-GAKKRRQRRR (SEQ ID NO: 52) X-AKKRRQRRR (SEQ ID NO: 53)X-GRKARRQRRR (SEQ ID NO: 54) X-RKARRQRRR (SEQ ID NO: 55)X-GRKKARQRRR (SEQ ID NO: 56) X-RKKARQRRR (SEQ ID NO: 57)X-GRKKRRQARR (SEQ ID NO: 58) X-RKKRRQARR (SEQ ID NO: 59)X-GRKKRRQRAR (SEQ ID NO: 60) X-RKKRRQRAR (SEQ ID NO: 61)X-RRPRRPRRPRR (SEQ ID NO: 62) X-RRARRARRARR (SEQ ID NO: 63)X-RRRARRRARR (SEQ ID NO: 64) X-RRRPRRRPRR (SEQ ID NO: 65)X-RRPRRPRR (SEQ ID NO: 66) X-RRARRARR (SEQ ID NO: 67)

X can represent a free amino terminus, one or more amino acids, or aconjugated moiety. Internalization peptides can be used in inverso orretro or inverso retro form with or without the linked peptide orpeptidomimetic being in such form.

Internalization peptides can be attached to pharmacological agents byconventional methods. For example, the agents can be joined tointernalization peptides by chemical linkage, for instance via acoupling or conjugating agent. Numerous such agents are commerciallyavailable and are reviewed by Wong, Chemistry of Protein Conjugation andCross-Linking, CRC Press (1991). Some examples of cross-linking reagentsinclude J-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) orN,N′-(1,3-phenylene) bismaleimide; N,N′-ethylene-bis-(iodoacetamide) orother such reagent having 6 to 11 carbon methylene bridges (whichrelatively specific for sulfhydryl groups); and1,5-difluoro-2,4-dinitrobenzene (which forms irreversible linkages withamino and tyrosine groups). Other cross-linking reagents includep,p′-difluoro-m,m′-dinitrodiphenylsulfone (which forms irreversiblecross-linkages with amino and phenolic groups); dimethyl adipimidate(which is specific for amino groups); phenol-1,4-disulfonylchloride(which reacts principally with amino groups); hexamethylenediisocyanateor diisothiocyanate, or azophenyl-p-diisocyanate (which reactsprincipally with amino groups); glutaraldehyde (which reacts withseveral different side chains) and disdiazobenzidine (which reactsprimarily with tyrosine and histidine).

For pharmacological agents that are peptides attachment to aninternalization peptide can be achieved by generating a fusion proteincomprising the peptide sequence fused, preferably at its N-terminus, toan internalization peptide.

Pharmacologic peptides, optionally fused to tat peptides, can besynthesized by solid phase synthesis or recombinant methods.Peptidomimetics can be synthesized using a variety of procedures andmethodologies described in the scientific and patent literature, e.g.,Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley &Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby(1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers.3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234.

IV. Inflammatory Response to Internalization Peptides

The present inventors have found that internalization peptides such astat have capacity to induce an inflammatory response on administrationto a subject. The inflammatory response is usually detectable within 1,5, 10, 20, 30, or 60 min of administering the peptide, but typicallydisappears within 24 hr of administration of the peptide (assuming thepeptide is not readministered). The inflammatory response isdose-dependent. The inflammatory response typically recurs at similarintensity on readministering the peptide. One aspect of the inflammatoryresponse is often a transient decrease in blood pressure occurringwithin a period of about 0-30 min after administering theinternalization peptide.

The inflammatory response is characterized by a degranulation of mastcells and consequent release of histamine and other mediators ofinflammation, such as chemokines, cytokines, leukotrienes, lipases,proteases, kinins, cytokines, arachidonic acid derivatives such asprostaglandins, interleukins, and/or nitric oxide (see FIG. 12). Thehistamine and/or other released mediators of inflammation give rise to anumber of symptoms of inflammation including redness of the skin, heat,swelling, hypotension and/or reduced pulse. Histamine release can alsoresult in vasodilation, hypotension, bronchoconstriction, smooth muscleactivation, separation of endothelial cells (responsible for hives),pain, itching, increased capillary permeability, glandularhypersecretion, smooth muscle spasm, and/or tissue infiltration ofinflammatory cells, as well as gastric acid secretion, and decreasedrelease of neurotransmitters such as histamine, acetylcholine,norepinephrine, and serotonin. Detection of any of these sequelae,particularly easily measurable ones, such as hypotension or a skin rash,such as hives, can be used as an indicator of mast cell degranulation.

V. Anti-Inflammatory Agents

A wide variety of anti-inflammatory agents are readily available toinhibit one or more aspects of the type of inflammatory response notedabove (see, e.g., U.S. Pat. No. 6,204,245, incorporated by reference).

A preferred class of anti-inflammatory agent is mast cell degranulationinhibitors. This class of compounds includes cromolyn(5,5′-(2-hydroxypropane-1,3-diyl)bis(oxy)bis(4-oxo-4H-chromene-2-carboxylicacid) (also known as cromoglycate), and2-carboxylatochromon-5′-yl-2-hydroxypropane derivatives such asbis(acetoxymethyl), disodium cromoglycate, nedocromil(9-ethyl-4,6-dioxo-10-propyl-6,9-dihydro-4H-pyrano[3,2-g]quinoline-2,8-dicarboxylicacid) and tranilast(2-{[(2E)-3-(3,4-dimethoxyphenyl)prop-2-enoyl]amino}), and lodoxamide(2-[2-chloro-5-cyano-3-(oxaloamino)anilino]-2-oxoacetic acid). Referenceto a specific compound includes pharmaceutically acceptable salts of thecompound Cromolyn is readily available in formulations for nasal, oral,inhaled or intravenous administration. Although practice of theinvention is not dependent on an understanding of mechanism, it isbelieved that these agents act at an early stage of inflammatoryresponse induced by an internalization peptide and are thus mosteffective at inhibiting development of its sequelae including atransient reduction in blood pressure. Other classes ofanti-inflammatory agent discussed below serve to inhibit one or moredownstream events resulting from mast cell degranulation, such asinhibiting histamine from binding to an H1 or H2 receptor, but may notinhibit all sequelae of mast cell degranulation or may require higherdosages or use in combinations to do so. Table 8 below summarizes thenames, chemical formulate and FDA status of several mast celldegranulation inhibitors that can be used with the invention.

TABLE 8 FDA Drug Name Alternative Names Chemical Formula statusAzelastine Astelin, Optivar 4-[(4-chlorophenyl)methyl]-2- Approved(1-methylazepan-4- yl)phthalazin-1-one Bepotastine Bepotastine besilate,Betotastine 4-[4-[(4-chlorophenyl)-pyridin- Approved besilate,TAU-284DS, bepotastine 2-ylmethoxy]piperidin-1- yl]butanoic acidChlorzoxazone Biomioran, EZE-DS, Escoflex, 5-chloro-3H-1,3-benzoxazol-2-Approved Flexazone, Mioran, Miotran, one Myoflexin, Myoflexine, Neoflex,Paraflex, Parafon Forte Dsc, Pathorysin, Relaxazone, Remular, Remular-S,Solaxin, Strifon Forte Dsc, Usaf Ma-10 Cromolyn Cromoglycate,Chromoglicate, 5-[3-(2-carboxy-4- Approved Chromoglicic Acid, Aarane,oxochromen-6-yl)oxy-2- Alercom, Alerion, Allergocrom, hydroxypropoxy]-4-ApoCromolyn, Children't oxochromene-2-carboxylic acid Nasalcrom,Colimune, Crolom, Cromolyn Nasal Solution, Cromoptic, Cromovet, Fivent,Gastrocrom, Gastrofrenal, GenCromoglycate, Inostral, Intal, Intal,Inhaler, Intal, Syncroner, Introl, Irtan, Lomudal, Lomupren, Lomusol,Lomuspray, Nalcrom, Nalcron, Nasalcrom, Nasmil, Opticrom, Opticron,Rynacrom, Sofro, Vistacrom, Vividrin Epinastine Elestat C16H15N3, CAS80012-43-7 Approved Isoproterenol Aerolone, Aleudrin, Aleudrine,4-[1-hydroxy-2-(propan-2- Approved Aludrin, Aludrine, Asiprenol,ylamino)ethyl]benzene-1,2-diol Asmalar, Assiprenol, Bellasthman,Bronkephrine, Euspiran, Isadrine, Isonorene, Isonorin, Isorenin,Isuprel, Isuprel Mistometer, Isupren, Medihaler- Iso, NeoEpinine,Neodrenal, Norisodrine, m Norisodrine, Aerotrol, Novodrin, Proternol,Respifral, Saventrine, Vapo-Iso Ketotifen Zaditor C19H19NOS, CAS34580-14-8 Approved Lodoxamide Alomide N,N′-(2-chloro-5-cyano-m-Approved (lodoxamide phenylene)dioxamic acid tromethamine) tromethaminesalt Nedocromil Alocril, Nedocromil 9-ethyl-4,6-dioxo-10- Approved[USAN:BAN:INN], Tilade propylpyrano[5,6-g]quinoline- 2,8-dicarboxylicacid Olopatadine Olopatadine Hydrochloride 2-[(11Z)-11-(3- ApprovedPatanol dimethylaminopropylidene)- 6H-benzo[c][2]benzoxepin-2- yl]aceticacid Pemirolast Alamast 9-methyl-3-(2H-tetrazol-5- Approvedyl)pyrido[2,1-b]pyrimidin-4- one Pirbuterol Maxair6-[2-(tert-butylamino)-1- Approved hydroxyethyl]-2-(hydroxymethyl)pyridin-3-ol

Another class of anti-inflammatory agent is anti-histamine compounds.Such agents inhibit the interaction of histamine with its receptorsthereby inhibiting the resulting sequelae of inflammation noted above.Many anti-histamines are commercially available, some over the counter.Examples of anti-histamines are azatadine, azelastine, burfroline,cetirizine, cyproheptadine, doxantrozole, etodroxizine, forskolin,hydroxyzine, ketotifen, oxatomide, pizotifen, proxicromil,N,N′-substituted piperazines or terfenadine. Anti-histamines vary intheir capacity to block anti-histamine in the CNS as well as peripheralreceptors, with second and third generation anti-histamines havingselectivity for peripheral receptors. Acrivastine, Astemizole,Cetirizine, Loratadine, Mizolastine, Levocetirizine, Desloratadine, andFexofenadine are examples of second and third generationanti-histamines. Anti-histamines are widely available in oral andtopical formulations. Some other anti-histamines that can be used aresummarized in Table 9 below.

TABLE 9 FDA Drug Name Alternative Names Chemical Formula statusKetotifen Ketotifen, Zaditor C19H19NOS Approved fumarate MequitazineButix, Instotal, Kitazemin, 10-(1-azabicyclo[2.2.2]octan-8- ApprovedMetaplexan, Mircol, Primalan, ylmethyl)phenothiazine Vigigan, Virginan,Zesulan Dexbrompheniramine Ilvan (3S)-3-(4-bromophenyl)-N,N- Approveddimethyl-3-pyridin-2-ylpropan- 1-amine Methdilazine Bristaline, Dilosyn,Disyncram, 10-[(1-methylpyrrolidin-3- Approved Disyncran, Tacaryl,Tacaryl yl)methyl]phenothiazine hydrochloride, Tacazyl, TacrylChlorpheniramine Aller-Chlor, Allergican, 3-(4-chlorophenyl)-N,N-Approved Allergisan, Antagonate, Chlo- dimethyl-3-pyridin-2-ylpropan-Amine, Chlor-Trimeton, Chlor- 1-amine Trimeton Allergy, Chlor- TrimetonRepetabs, Chlor- Tripolon, Chlorate, Chloropiril, Cloropiril, Efidac 24Chlorpheniramine Maleate, Gen- Allerate, Haynon, Histadur, Kloromin,Mylaramine, Novo- Pheniram, Pediacare Allergy Formula, Phenetron,Piriton, Polaramine, Polaronil, Pyridamal 100, Telachlor, TeldrinBromopheniramine Bromfed, Bromfenex, Dimetane, 3-(4-bromophenyl)-N,N-Approved Veltane dimethyl-3-pyridin-2-ylpropan- 1-amine TerbutalineBrethaire, Brethine, Brican, 5-[2-(tert-butylamino)-1- ApprovedBricanyl, Bricar, Bricaril, Bricyn hydroxyethyl]benzene-1,3-diolpimecrolimus Elidel (3S,4R,5S,8R,9E,12S,14S,15R, Approved16S,18R,19R,26aS)-3-{(E)-2- as [(1R,3R,4S)-4-Chloro-3- topical,methoxycyclohexyl]-1- Investiga- methylvinyl} -8-ethyl- tional as5,6,8,11,12,13,14,15,16,17,18,19, oral 24,25,26,26a-hexadecahydro-5,19-dihydroxy-14,16- dimethoxy-4,10,12,18- tetramethyl-15,19-epoxy-3H-pyrido[2,l- c][1,4]oxaazacyclotricosine- 1,7,20,21(4H,23H)-tetrone

Another class of anti-inflammatory agent useful in inhibiting theinflammatory response is corticosteroids. These compounds aretranscriptional regulators and are powerful inhibitors of theinflammatory symptoms set in motion by release of histamine and othercompounds resulting from mast cell degranulation. Examples ofcorticosteroids are Cortisone, Hydrocortisone (Cortef), Prednisone(Deltasone, Meticorten, Orasone), Prednisolone (Delta-Cortef, Pediapred,Prelone), Triamcinolone (Aristocort, Kenacort), Methylprednisolone(Medrol), Dexamethasone (Decadron, Dexone, Hexadrol), and Betamethasone(Celestone). Corticosteriods are widely available in oral, intravenousand topical formulations.

Nonsteroidal anti-inflammatory drugs (NSAIDs) can also be used. Suchdrugs include aspirin compounds (acetylsalicylates), non-aspirinsalicylates, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen,ibuprofen, indomethacin, ketoprofen, meclofenamate, naproxen, naproxensodium, phenylbutazone, sulindac, and tometin. However, theanti-inflammatory effects of such drugs are less effective than those ofanti-histamines or corticosteroids.

Stronger anti-inflammatory drugs such as azathioprine, cyclophosphamide,leukeran, and cyclosporine can also be used but are not preferredbecause they are slower acting and/or associated with side effects.Biologic anti-inflammatory agents, such as Tysabri® or Humira®, can alsobe used but are not preferred for the same reasons.

Different classes of drugs can be used in combinations in inhibiting aninflammatory response. A preferred combination is a mast celldegranulation inhibitor and an anti-histamine.

VI. Conjugation

The inflammatory response inducible by an internalization peptide canalternatively (or additionally) be reduced by linking theinternalization peptide to biotin or similar molecule to form aconjugate. The conjugate retains an ability to facilitate uptake of alinked pharmacologic agent into cells into cells but induces a reducedinflammatory response compared to the same internalization peptidewithout the biotin. Conjugated internalization peptides can be screenedto confirm desired uptake and lack of (or decrease in) a resultingimmune response.

Alternatives to biotin that can be used to form conjugates of aninternalization peptide include acetyl, benzoyl, alkyl group(aliphatic), pyroglutamate, alkyl group with cycloalkyl group at theend, biotin with alkyl spacer, (5,6)-FAM. The biotin or other moleculecan be linked to the internationalization peptide through an amidechemistry, sulphamide chemistry, sulphone chemistry, and/or alkylationchemistry.

VII. Patients Amenable to Treatment/Prophylaxis

A broad range of patients are amenable to treatment by the methods ofinvention as exemplified by the pharmacologic agents and associatedconditions listed in Table 1. The methods are of particular use in suchpatients having a condition that would exacerbate any inflammationresulting from an internalization peptide, for example, a patientsuffering from hypertension, elevated pulse or other signs or symptomsof inflammation. The methods are also particularly useful in methods oftreatment requiring a high dose of pharmacological agent linked tointernalization peptide. Strictly it is the dose of the internalizationpeptide rather than the linked pharmacological agent that determinespresence and extent of the inflammatory response, if any. However, thedoes of internalization peptide is of course determined by the dose ofthe linked pharmacological agent. For example, an inflammatory responsemay become noticeable at a dose of greater than 1.5 mg/kginternalization peptide). In treatment of some diseases, the effectivedose of pharmacologic agent and consequently linked internalizationpeptide is too low to induce an inflammatory response in most patients.Nevertheless, the sensitivity of individual patients to an inflammatoryresponse can vary, and treatment with a mild anti-inflammatory agent,such as a histamine, can still be a worthwhile precaution.

One class of patients of particular interest is those having or at riskof a disease or condition characterized by excitotoxicity. Such diseasesand conditions include stroke, epilepsy, hypoxia, traumatic injury tothe CNS not associated with stroke such as traumatic brain injury andspinal cord injury, Alzheimer's disease and Parkinson's disease. Suchconditions also include patients undergoing surgery that affects or mayaffect a vessel (e.g., jugular vein or carotid artery) supplying orremoving blood to or from the brain, particularly patients undergoingneurosurgery, such as endovascular surgery to repair an aneurysm orendovascular surgery to a blood vessel supplying a limb, spinal cord,retina or kidney (see 61/185,989 filed Jun. 10, 2009 and filedherewith). Such repair can be effected for example by inserting a stentor coil into the blood vessel subject to the aneurysm. The methods ofthe invention are suitable for treating both male and female patientshaving or at risk of such diseases and conditions.

A stroke is a condition resulting from impaired blood flow in the CNSregardless of cause. Potential causes include embolism, hemorrhage andthrombosis. Some neuronal cells die immediately as a result of impairedblood flow. These cells release their component molecules includingglutamate, which in turn activates NMDA receptors, which raiseintracellular calcium levels, and intracellular enzyme levels leading tofurther neuronal cell death (the excitotoxicity cascade). The death ofCNS tissue is referred to as infarction. Infarction Volume (i.e., thevolume of dead neuronal cells resulting from stroke in the brain) can beused as an indicator of the extent of pathological damage resulting fromstroke. The symptomatic effect depends both on the volume of aninfarction and where in the brain it is located. Disability index can beused as a measure of symptomatic damage, such as the Rankin StrokeOutcome Scale (Rankin, Scott Med J; 2:200-15 (1957)) and the BarthelIndex. The Rankin Scale is based on assessing directly the globalconditions of a patient as follows.

TABLE 4 0 No symptoms at all 1 No significant disability despitesymptoms; able to carry out all usual duties and activities. 2 Slightdisability; unable to carry out all previous activities but able to lookafter own affairs without assistance. 3 Moderate disability requiringsome help, but able to walk without assistance 4 Moderate to severedisability; unable to walk without assistance and unable to attend toown bodily needs without assistance. 5 Severe disability; bedridden,incontinent, and requiring constant nursing care and attention.

The Barthel Index is based on a series of questions about the patient'sability to carry out 10 basic activities of daily living resulting in ascore between 0 and 100, a lower score indicating more disability(Mahoney et al., Maryland State Medical Journal 14:56-61 (1965)).

Alternatively stroke severity/outcomes can be measured using the NIHstroke scale, available at world wide web nindsnih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf.

The scale is based on the ability of a patient to carry out 11 groups offunctions that include assessments of the patient's level ofconsciousness, motor, sensory and language functions.

An ischemic stroke refers more specifically to a type of stroke thatcaused by blockage of blood flow to the brain. The underlying conditionfor this type of blockage is most commonly the development of fattydeposits lining the vessel walls. This condition is calledatherosclerosis. These fatty deposits can cause two types ofobstruction. Cerebral thrombosis refers to a thrombus (blood clot) thatdevelops at the clogged part of the vessel “Cerebral embolism” refersgenerally to a blood clot that forms at another location in thecirculatory system, usually the heart and large arteries of the upperchest and neck. A portion of the blood clot then breaks loose, entersthe bloodstream and travels through the brain's blood vessels until itreaches vessels too small to let it pass. A second important cause ofembolism is an irregular heartbeat, known as arterial fibrillation. Itcreates conditions in which clots can form in the heart, dislodge andtravel to the brain. Additional potential causes of ischemic stroke arehemorrhage, thrombosis, dissection of an artery or vein, a cardiacarrest, shock of any cause including hemorrhage, and iatrogenic causessuch as direct surgical injury to brain blood vessels or vessels leadingto the brain or cardiac or pulmonary surgery. Ischemic stroke accountsfor about 83 percent of all cases of stroke.

Transient ischemic attacks (TIAs) are minor or warning strokes. In aTIA, conditions indicative of an ischemic stroke are present and thetypical stroke warning signs develop. However, the obstruction (bloodclot) occurs for a short time and tends to resolve itself through normalmechanisms. Patients undergoing heart, pulmonary or neuro-surgery are atparticular risk of transient cerebral ischemic attack.

Hemorrhagic stroke accounts for about 17 percent of stroke cases. Itresults from a weakened vessel that ruptures and bleeds into thesurrounding brain. The blood accumulates and compresses the surroundingbrain tissue. The two general types of hemorrhagic strokes areintracerebral hemorrhage and subarachnoid hemorrhage. Hemorrhagic strokeresult from rupture of a weakened blood vessel. Potential causes ofrupture from a weakened blood vessel include a hypertensive hemorrhage,in which high blood pressure causes a rupture of a blood vessel, oranother underlying cause of weakened blood vessels such as a rupturedbrain vascular malformation including a brain aneurysm, arteriovenousmalformation (AVM) or cavernous malformation. Hemorrhagic strokes canalso arise from a hemorrhagic transformation of an ischemic stroke whichweakens the blood vessels in the infarct, or a hemorrhage from primaryor metastatic tumors in the CNS which contain abnormally weak bloodvessels. Hemorrhagic stroke can also arise from iatrogenic causes suchas direct surgical injury to a brain blood vessel. An aneurysm is aballooning of a weakened region of a blood vessel. If left untreated,the aneurysm continues to weaken until it ruptures and bleeds into thebrain. An arteriovenous malformation (AVM) is a cluster of abnormallyformed blood vessels. A cavernous malformation is a venous abnormalitythat can cause a hemorrhage from weakened venous structures. Any one ofthese vessels can rupture, also causing bleeding into the brain.Hemorrhagic stroke can also result from physical trauma. Hemorrhagicstroke in one part of the brain can lead to ischemic stroke in anotherthrough shortage of blood lost in the hemorrhagic stroke.

VIII. Delivery of Pharmacological Agent with an Anti-Inflammatory Agent

In methods in which a pharmacological agent linked to an internalizationpeptide is administered with an anti-inflammatory agent, the twoentities are administered sufficiently proximal in time that theanti-inflammatory agent can inhibit an inflammatory response inducibleby the internalization peptide. The anti-inflammatory agent can beadministered before, at the same time as or after the pharmacologicagent, but is preferably administered before. The preferred time dependsin part on the pharmacokinetics and pharmacodynamics of theanti-inflammatory agent. The anti-inflammatory agent can be administeredat an interval before the pharmacologic agent such that theanti-inflammatory agent is near maximum serum concentration at the timethe pharmacologic agent is administered. Typically, theanti-inflammatory agent is administered between 6 hours before thepharmacological agent and one hour after. For example, theanti-inflammatory agent can be administered between 1 hour before and 30min after the pharmacological agent. Preferably the anti-inflammatoryagent is administered between 30 minutes before and 15 minutes after thepharmacologic agent, and more preferably within 15 minutes before andthe same time as the pharmacological agent. In some methods, theanti-inflammatory agent is administered before the pharmacological agentwithin a period of 15, 10 or 5 minutes before the pharmacological agentis administered. In some methods, the agent is administered 1-15, 1-10or 1-5 minutes before the pharmacological agent.

When administration of an agent is not instantaneous, such as withintravenous infusion, the anti-inflammatory agent and pharmacologicalagent are considered to be administered at the same time if theirperiods of administration are co-extensive or overlap. Time periods ofadministration before administration start from the beginning of itsadministration. Time periods after administration start from the end ofits administration. Time periods referring to the administration of theanti-inflammatory agent refer to the beginning of its administration.

When an anti-inflammatory agent is said to be able to inhibit theinflammatory response of a pharmacological agent linked to aninternalization peptide what is meant is that the two are administeredsufficiently proximate in time that the anti-inflammatory agent wouldinhibit an inflammatory response inducible by the pharmacological agentlinked to the internalization peptide if such a response occurs in aparticular patient, and does not necessarily imply that such a responseoccurs in that patient. Some patients are treated with a dose ofpharmacological agent linked to an internalization peptide that isassociated with an inflammatory response in a statistically significantnumber of patients in a controlled clinical or nonclinical trial. It canreasonably be assumed that a significant proportion of such patientsalthough not necessarily all develop an anti-inflammatory response tothe pharmacological agent linked to the internalization peptide. In somepatients, signs or symptoms of an inflammatory response to thepharmacological agent linked to the internalization peptide are detectedor detectable.

In clinical treatment of an individual patient, it is not usuallypossible to compare the inflammatory response from a pharmacologicalagent linked to an internalization peptide in the presence and absenceof an anti-inflammatory agent. However, it can reasonably be concludedthat the anti-inflammatory agent inhibits an anti-inflammatory responseinducible by the peptide if significant inhibition is seen under thesame or similar conditions of co-administration in a controlled clinicalor pre-clinical trial. The results in the patient (e.g., blood pressure,heart rate, hives) can also be compared with the typical results of acontrol group in a clinical trial as an indicator of whether inhibitionoccurred in the individual patient. Usually, the anti-inflammatory agentis present at a detectable serum concentration at some point within thetime period of one hour after administration of the pharmacologic agent.The pharmacokinetics of many anti-inflammatory agents is widely knownand the relative timing of administration of the anti-inflammatory agentcan be adjusted accordingly. The anti-inflammatory agent is usuallyadministered peripherally, i.e., segregated by the blood brain barrierfrom the brain. For example, the anti-inflammatory agent can beadministered orally, nasally, intravenously or topically depending onthe agent in question. If the anti-inflammatory agent is administered atthe same time as the pharmacologic agent, the two can be administered asa combined formulation or separately.

In some methods, the anti-inflammatory agent is one that does not crossthe blood brain barrier when administered orally or intravenously atleast in sufficient amounts to exert a detectable pharmacologicalactivity in the brain. Such an agent can inhibit mast cell degranulationand its sequelae resulting from administration of the pharmacologicalagent in the periphery without itself exerting any detectabletherapeutic effects in the brain. In some methods, the anti-inflammatoryagent is administered without any co-treatment to increase permeabilityof the blood brain barrier or to derivatize or formulate theanti-inflammatory agent so as to increase its ability to cross the bloodbrain barrier. However, in other methods, the anti-inflammatory agent,by its nature, derivatization, formulation or route of administration,may by entering the brain or otherwise influencing inflammation in thebrain, exert a dual effect in suppressing mast-cell degranulation and/orits sequelae in the periphery due to an internalization peptide andinhibiting inflammation in the brain. Strbian et al., WO 04/071531 havereported that a mast cell degranulation inhibitor, cromoglycate,administered i.c.v. but not intravenously has direct activity ininhibiting infarctions in an animal model.

In some methods, the patient is not also treated with the sameanti-inflammatory agent co-administered with the pharmacological agentin the day, week or month preceding and/or following co-administrationwith pharmacological agent. In some methods, if the patient is otherwisebeing treated with the same anti-inflammatory agent co-administered withthe pharmacological agent in a recurring regime (e.g., same amount,route of delivery, frequency of dosing, timing of day of dosing), theco-administration of the anti-inflammatory agent with thepharmacological agent does not comport with the recurring regime in anyor all of amount, route of delivery, frequency of dosing or time of dayof dosing. In some methods, the patient is not known to be sufferingfrom an inflammatory disease or condition requiring administration ofthe anti-inflammatory agent co-administered with the pharmacologicalagent in the present methods. In some methods, the patient is notsuffering from asthma or allergic disease treatable with a mast celldegranulation inhibitor. In some methods, the anti-inflammatory agentand pharmacological agent are each administered once and only oncewithin a window as defined above, per episode of disease, an episodebeing a relatively short period in which symptoms of disease are presentflanked by longer periods in which symptoms are absent or reduced.

The anti-inflammatory agent is administered in a regime of an amount,frequency and route effective to inhibit an inflammatory response to aninternalization peptide under conditions in which such an inflammatoryresponse is known to occur in the absence of the anti-inflammatory. Aninflammatory response is inhibited if there is any reduction in signs orsymptoms of inflammation as a result of the anti-inflammatory agent.Symptoms of the inflammatory response can include redness, rash such ashives, heat, swelling, pain, tingling sensation, itchiness, nausea,rash, dry mouth, numbness, airway congestion. The inflammatory responsecan also be monitored by measuring signs such as blood pressure, orheart rate. Alternatively, the inflammatory response can be assessed bymeasuring plasma concentration of histamine or other compounds releasedby mast cell degranulation. The presence of elevated levels of histamineor other compounds released by mast cell degranulation, reduced bloodpressure, skin rash such as hives, or reduced heart rate are indicatorsof mass cell degranulation. As a practical matter, the doses, regimesand routes of administration of most of the anti-inflammatory agentsdiscussed above are available in the Physicians' Desk Reference and/orfrom the manufacturers, and such anti-inflammatories can be used in thepresent methods consistent with such general guidance.

IX. Methods of Treatment/Prophylaxis

a) Methods of Treatment

A chimeric agent comprising a pharmacologic agent attached to aninternalization peptide is administered in an amount, frequency androute of administration effective to cure, reduce or inhibit furtherdeterioration of at least one sign or symptom of a disease in a patienthaving the disease being treated. A therapeutically effective amountmeans an amount of chimeric agent sufficient significantly to cure,reduce or inhibit further deterioration of at least one sign or symptomof the disease or condition to be treated in a population of patients(or animal models) suffering from the disease treated with a chimericagent of the invention relative to the damage in a control population ofpatients (or animal models) suffering from that disease or condition whoare not treated with a chimeric agent of the invention. The amount isalso considered therapeutically effective if an individual treatedpatient achieves an outcome more favorable than the mean outcome in acontrol population of comparable patients not treated by methods of theinvention. A therapeutically effective regime involves theadministration of a therapeutically effective dose at a frequency androute of administration needed to achieve the intended purpose.

For a patient suffering from stroke or other ischemic condition, thechimeric agent is administered in a regime comprising an amountfrequency and route of administration effective to reduce the damagingeffects of stroke or other ischemic condition. When the conditionrequiring treatment is stroke, the outcome can be determined byinfarction volume or disability index, and a dosage is consideredtherapeutically effective if an individual treated patient shows adisability of two or less on the Rankin scale and 75 or more on theBarthel scale, or if a population of treated patients shows asignificantly improved (i.e., less disability) distribution of scores ona disability scale than a comparable untreated population, see Lees etal., N Engl J Med 2006; 354:588-600. A single dose of chimeric agent isusually sufficient for treatment of stroke.

b) Methods of Prophylaxis

The invention also provides methods and compositions for the prophylaxisof a disorder in a subject at risk of that disorder. Usually such asubject has an increased likelihood of developing the disorder (e.g., acondition, illness, disorder or disease) relative to a controlpopulation. The control population for instance can comprise one or moreindividuals selected at random from the general population (e.g.,matched by age, gender, race and/or ethnicity) who have not beendiagnosed or have a family history of the disorder. A subject can beconsidered at risk for a disorder if a “risk factor” associated withthat disorder is found to be associated with that subject. A risk factorcan include any activity, trait, event or property associated with agiven disorder, for example, through statistical or epidemiologicalstudies on a population of subjects. A subject can thus be classified asbeing at risk for a disorder even if studies identifying the underlyingrisk factors did not include the subject specifically. For example, asubject undergoing heart surgery is at risk of transient cerebralischemic attack because the frequency of transient cerebral ischemicattack is increased in a population of subjects who have undergone heartsurgery as compared to a population of subjects who have not.

Other common risk factors for stroke include age, family history,gender, prior incidence of stroke, transient ischemic attack or heartattack, high blood pressure, smoking, diabetes, carotid or other arterydisease, atrial fibrillation, other heart diseases such as heartdisease, heart failure, dilated cardiomyopathy, heart valve diseaseand/or congenital heart defects; high blood cholesterol, and diets highin saturated fat, trans fat or cholesterol.

Pharmacological agents linked to an internalization peptide areadministered to patients at risk of a disease but not yet having thedisease in an amount, frequency and route sufficient to prevent, delayor inhibit development of at least one sign or symptom of the disease. Aprophylactically effective amount means an amount of chimeric agentsufficient significantly to prevent, inhibit or delay at least one signor symptom of the disease in a population of patients (or animal models)at risk of the disease relative treated with the agent compared to acontrol population of patients (or animal models) at risk of the diseasenot treated with a chimeric agent of the invention. The amount is alsoconsidered prophylactically effective if an individual treated patientachieves an outcome more favorable than the mean outcome in a controlpopulation of comparable patients not treated by methods of theinvention. A prophylactically effective regime involves theadministration of a prophylactically effective dose at a frequency androute of administration needed to achieve the intended purpose. Forprophylaxis of stroke in a patient at imminent risk of stroke (e.g., apatient undergoing heart surgery), a single dose of chimeric agent isusually sufficient.

X. Pharmaceutical Compositions, Dosages, and Routes of Administration

The chimeric agents of the invention can be administered in the form ofa pharmaceutical composition. Pharmaceutical compositions are typicallymanufactured under GMP conditions. Pharmaceutical compositions can beprovided in unit dosage form (i.e., the dosage for a singleadministration). Pharmaceutical compositions can be manufactured bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. For example, lyophilized chimeric agents of the invention canbe used in the formulations and compositions described below.

Pharmaceutical compositions can be formulated in conventional mannerusing one or more physiologically acceptable carriers, diluents,excipients or auxiliaries that facilitate processing of chimeric agentsinto preparations which can be used pharmaceutically. Proper formulationis dependent on the route of administration chosen.

Some pharmaceutical composition are a co-formulation of an active agentand an anti-inflammatory agent as described above for simultaneousadministration of the active agent and anti-inflammatory agent. Suchco-formulations typically have the active agents dissolved in solution,although the active agents can also by co-lyophilized or individuallylyophilized and mixed, and then reformulated before use. Whether mixedfrom solutions or by reconstituting a lyophilysate, the formulation ispreferably substantially free of visible particles on formation (i.e.,less than 10%, 5% or 1% of each active agent is in particulate form).The formulation preferably remains substantially free of visibleparticles on storage for at least a week, a month or a year. Theformulation can be stored in cold liquid form (in a refrigerator atabout 4 degrees C. or can be stored in frozen form.

One example of such a composition is a co-formulation of Tat-NR2B9c withlodoxamide. The two active agents can be formulated in an aqueoussolution at a range of concentrations of the active agents. For example,the concentration of Tat-NR2B9c can range from about 1-30 mg/ml and thatof lodoxamide from 0.1 to 5 mg/ml. The co-formulation can also contain atonicity agent (e.g., NaCl), hydrochloric acid or sodium hydroxide toadjust pH, a buffer a preservative, and various other excipients used inthe commercial preparation of lodoxamide, such as mannitol,hydroxypropyl methylcellulose 2910, sodium citrate, citric acid, edetatedisodium, tyloxapol. The formulation can be prepared, for example,simply by mixing Tat-NR2B9c in saline with Alomide (lodoxamide), asfurther defined in the Examples and vortexing. The Tat-NR2B9c ispreferably at a concentration of 10-30 mg/ml or more preferably 20mg/ml. The NA-1 and lodoxamide are combined in a ratio of about 2:3 ormore preferably 1.89:3.11.

Administration can be parenteral, intravenous, oral, subcutaneous,intra-arterial, intracranial, intrathecal, intraperitoneal, topical,intranasal or intramuscular. Intravenous administration is preferred.

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic. For injection, chimeric agents canbe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological saline or acetate buffer (to reduce discomfort at the siteof injection). The solution can contain formulatory agents such assuspending, stabilizing and/or dispersing agents.

Alternatively the chimeric agents can be in powder form for constitutionwith a suitable vehicle, e.g., sterile pyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. This route ofadministration can be used to deliver the compounds to the nasal cavityor for sublingual administration.

For oral administration, the chimeric agents can be formulated withpharmaceutically acceptable carriers as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a patient to be treated. For oral solid formulationssuch as, for example, powders, capsules and tablets, suitable excipientsinclude fillers such as sugars, such as lactose, sucrose, mannitol andsorbitol; cellulose preparations such as maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP); granulating agents; and binding agents. Ifdesired, disintegrating agents can be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. If desired, solid dosage forms can be sugar-coated orenteric-coated using standard techniques. For oral liquid preparationssuch as, for example, suspensions, elixirs and solutions, suitablecarriers, excipients or diluents include water, glycols, oils, alcohols.Additionally, flavoring agents, preservatives, coloring agents and thelike can be added.

In addition to the formulations described previously, the chimericagents can also be formulated as a depot preparation. Such long actingformulations can be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds can be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Alternatively, other pharmaceutical delivery systems can be employed.Liposomes and emulsions can be used to deliver chimeric agents. Certainorganic solvents such as dimethylsulfoxide also can be employed,although usually at the cost of greater toxicity. Additionally, thecompounds can be delivered using a sustained-release system, such assemipermeable matrices of solid polymers containing the therapeuticagent.

Sustained-release capsules can, depending on their chemical nature,release the chimeric agents for a few weeks up to over 100 days.Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization canbe employed.

As the chimeric agents of the invention can contain charged side chainsor termini, they can be included in any of the above-describedformulations as the free acids or bases or as pharmaceuticallyacceptable salts. Pharmaceutically acceptable salts are those saltswhich substantially retain the biologic activity of the free bases andwhich are prepared by reaction with inorganic acids. Pharmaceuticalsalts tend to be more soluble in aqueous and other protic solvents thanare the corresponding free base forms.

Chimeric agents comprising an internalization peptide linked to apharmacologic agent can be used at the same or lower dosage on a molarbasis as the pharmacologic agent alone, and can be administered by thesame route as the pharmacologic agent alone, and for treatment of thesame disease(s) as the pharmacologic agent alone.

For treatment of stroke, preferred dosage ranges include 0.001 to 20μmol chimeric peptide or peptidomimetic per kg patient body weight,optionally 0.03 to 3 μmol chimeric peptide per kg patient body weight.In some methods, 0.1-20 μmol chimeric peptide or peptidomimetic per kgpatient body weight is administered. In some methods, 0.1-10 μmolchimeric peptide or peptidomimetic per kg patient body weight, morepreferably about 0.3 μmol chimeric peptide per kg patient body weight.In other instances, the dosages range is from 0.005 to 0.5 μmol chimericpeptide or peptidomimetic per kg patient body weight. Dosage per kg bodyweight can be converted from rats to humans by dividing by 6.2 tocompensate for different surface area to mass ratios. Dosages can beconverted from units of moles to grams by multiplying by the molarweight of a chimeric peptide or peptidomimetic. Suitable dosages ofchimeric peptides or peptidomimetics for use in humans can include 0.001to 5 mg/kg patient body weight, or 0.005 to 1 mg/kg patient body weightor 0.05 to 1 mg/kg, or 0.09 to 0.9 mg/kg. In absolute weight for a 75 kgpatient, these dosages translate to 0.075-375 mg, 0.375 to 75 mg or 3.75mg to 75 mg or 6.7 to 67 mg. Rounded to encompass variations in e.g.,patient weight, the dosage is usually within 0.05 to 500 mg, preferably0.1 to 100 mg, 0.5 to 50 mg, or 1-20 mg. Indicated dosages should beunderstood as including the margin of error inherent in the accuracywith which dosages can be measured in a typical hospital setting.

The co-administration of an anti-inflammatory agent with apharmacological agent is particularly useful when the pharmacologicalagent is administered at higher doses when mast cell degranulation ismost likely to ensue. For administration of the chimeric agentTat-NR2B9c to humans, an approximate dosage level when mast celldegranulation is likely to occur is a dose of greater than or equal to2.6 mg/kg. Thus, co-administration of an antiinflammatory agent isparticularly useful at a dose of Tat-NR2B9c of greater than or equal to2.6 mg/kg, 3 mg/kg, 5 mg/kg or 10 mg/kg. Usually the dosage is nothigher than 50 mg/kg.

Although lower dosages may be equally effective in many patients, use ofhigh dosages is advantageous in extremely acute diseases, such asstroke, in which if the first administration of a pharmacological agentis insufficient, there may be little opportunity for a second. Ofcourse, some variation is expected between individual patients in theprecise dosage at which onset of mast cell degranulation occurs.Therefore, it can also be useful to administer the anti-inflammatory asa precaution at lower dosages of Tat-NR2B9c in case mast celldegranulation occurs in a few patients with greater than normalsusceptibility. For example, the anti-inflammatory agent can beadministered at dosages of Tat-NR2B9c of greater or equal to 0.5 mg/kg,1 mg/mg or 2 mg/kg.

In some methods, in which a population of patients are treated, somepatients are administered an anti-inflammatory agent and some are notdepending on the dose of the pharmacological agent linked to theinternalization peptide with patients receiving a higher dose receivingthe anti-inflammatory agent. The transition point for administering ornot administering the anti-inflammatory agent for Tat-NR2B9c can be adose from about 1-3 mg/kg. For example, patients receiving 1 mg/kg orless are not administered the anti-inflammatory agent, patientsreceiving 3 mg/kg or greater are administered the anti-inflammatoryagent and patients receiving a dose of greater than 1 and less than 3mg/kg, may or may not be administered the anti-inflammatory. Of course,the above scheme is just an example, and a different transition pointcan be set. Also, all patients receiving a dose at or above thetransition point can be administered the anti-inflammatory and allpatients receiving a dose below the transition point may be treatedwithout the anti-inflammatory. Also, all patients can be administeredthe anti-inflammatory without regard to the dose of the pharmacologicalagent linked to the internalization peptide, as discussed above. Thedosages indicated above for the chimeric agent Tat-NR2B9c(YGRKKRRQRRRKLSSIESDV; SEQ ID NO:6) can also be used for close variantsof that agent in which one or a few amino acids are substituted,inserted or deleted and the molecular weight remains the same withinabout +/−25% However, in general, equivalent dosages of other agents forpurposes of determining when to administer an inflammatory agent can bedetermined by calculating the dose of that agent that delivers anequimolar amount of internalization peptide to a given dose ofTat-NR2B9c.

The amount of chimeric agent administered depends on the subject beingtreated, on the subject's weight, the severity of the affliction, themanner of administration and the judgment of the prescribing physician.The therapy can be repeated intermittently while symptoms are detectableor even when they are not detectable. The therapy can be provided aloneor in combination with other drugs. For treatment of stroke, the dose ofpharmacologic agent linked to an internalization peptide is usuallyadministered within 24 hours of onset of stroke, preferably within 6hours of onset of stroke.

XI. Kits

Kits are provided for carrying out the present methods. The kits includeone or more pharmacologic agents of interest, attached to aninternalization peptide. The internalization peptide can bebiotinylated, and/or the kit can contain an anti-inflammatory agent. Theinstant kit optionally contains means for administering thepharmacologic agents and/or anti-inflammatory agent. The kit can alsoinclude one or more buffers, additives, fillers or diluents. The kit canalso provide one or more printed instructions on the administration anddosage regimen to be followed.

XII. Screening Methods

A. Measuring Internalization Activity

Variants of the tat or other internalization peptide can be tested fortransport activity in an animal. Internalization peptides can be testedalone or when linked to an active agent, such an active peptide, e.g.,KLSSIESDV (SEQ ID NO:5). The internalization peptide, optionally linkedto an active agent, such as a peptide, is labeled, preferably with afluorescent label, such as dansyl chloride. The internalization peptideis then injected peripherally into an animal, such as a mouse.Intraperitoneal or intravenous injection is suitable, for example. Aboutan hour after injection, the mice are sacrificed, perfused with fixativesolution (3% paraformaldehyde, 0.25% glutaraldehyde, 10% sucrose, 10U/mL heparin in saline). Brains are then removed, frozen and sections.Sections are analyzed for fluorescence using a confocal microscope.Internalization activity is determined from fluorescence, optionallyrelative to positive and negative controls. A suitable positive controlis the standard tat peptide linked to the same active peptide (ifpresent) as the internalization peptide under test. A suitable negativecontrol is fluorescently labeled active peptide not linked to aninternalization peptide. Unlabelled vehicle can also be used as anegative control.

Similar experiments can be performed in cell culture to test variants oftat or other internalization peptide (see US20030050243). A variantfluorescently labeled tat peptide, optionally linked to an activepeptide is applied to a cortical neuronal culture. Uptake is determinedusing fluorescence microscopy over several minutes after application.Increased uptake can be determined relative to positive and negativecontrols as described for the experiments on uptake in an animal.

2. Measuring Activity in Treating Stroke

The activity of chimeric agents comprising a internalization peptidelinked to an agent can be tested in various animal models of stroke. Inone such model, in adult male Sprague-Dawley rats subjected to transientmiddle cerebral artery occlusion (MCAO) for 90 minutes by theintraluminal suture method (36,37). Animals are fasted overnight andinjected with atropine sulfate (0.5 mg/kg IP). After 10 minutesanesthesia is induced. Rats are orally intubated, mechanicallyventilated, and paralyzed with pancuronium bromide (0.6 mg/kg IV). Bodytemperature is maintained at 36.5-37.5° C., with a heating lamp.Polyethylene catheters in the femoral artery and vein are used tocontinuously record blood pressure and to sample blood for gas and pHmeasurements. Transient MCAO is achieved for 90 min by introducing apoly-L-lysine-coated 3-0 monofilament nylon suture (Harvard Apparatus)into the circle of Willis via the internal carotid artery, effectivelyoccluding the middle cerebral artery. This produces an extensiveinfarction encompassing the cerebral cortex and basal ganglia. Animalsare treated with either a chimeric agent under test or a negative orpositive control. Treatment can be either before or up to one hour afterinducing ischemia. A negative control can be vehicle. A positive controlcan be the Tat-NR2B9c peptide, YGRKKRRQRRRKLSSIESDV (SEQ ID NO:6),previously shown to be effective. The chimeric agent is delivered by asingle intravenous bolus injection 45 min prior to MCAO (3 nmoles/g).After administering compounds to the animals, infarction volume and/ordisability index are determined. Infarction volumes are usuallydetermined 24 hr post treatment but can be determined at a later timesuch as 3, 7, 14 or 60 days. Disability index can be monitored overtime, e.g., at 2 hr post treatment, 24 hr post treatment, one week andone month post treatment. Chimeric agents showing a statisticallysignificant reduction in infarction volume and/or disability indexrelative to control animals not treated with the compounds areidentified as having activity useful for practicing the methods of theinvention.

Similar experiments can be performed in animal subject to permanentischemia. Permanent ischemia of the middle cerebral artery pial vesselcan be carried out as described by Forder et al., Am J Physiol HeartCirc Physiol 288:H1989-H1996 (2005). In brief, the right ECA iscannulated with PE 10 polyethylene tubing. The skull is exposed via amidline incision, and a 6- to 8-mm cranial window is made over the rightsomatosensory cortex (2 mm caudal and 5 mm lateral to bregma). The pialarteries are visualized by injecting small boluses (10-20 μL) of thevital dye patent blue violet (10 mMol/L; Sigma) in normal saline, intothe ECA. The same three pial arteriolar MCA branches are electricallycauterized and dye injections are repeated to ensure the interruption offlow through the cauterized arterioles. The incision is then closed andthe animal returned to its cage and allowed free access to food andwater. This permanent ischemia model produces a highly reproduciblesmall infarction limited to the cortex underlying the coagulatedterminal pial arteries.

The left middle cerebral artery can be occluded by the intraluminalsuture method described by Longa, Stroke 20, 84-91 (1989). In brief, theleft common carotid artery (CCA) is exposed through a midline neckincision and is dissected free from surrounding nerves and fascia, fromits bifurcation to the base of the skull. The occipital artery branchesof the external carotid artery (ECA) are then isolated, and thesebranches dissected and coagulated. The ECA is dissected further distallyand coagulated along with the terminal lingual and maxillary arterybranches, which are then divided. The internal carotid artery (ICA) isisolated and separated from the adjacent vagus nerve, and thepterygopalatine artery is ligated close to its origin. The tip of a 4-cmlength of 3-0 monofilament nylon suture (Harvard Apparatus) is roundedby burning to achieve a tip diameter of 0.33-0.36 mm and tip length of0.5-0.6 mm and coated with poly-L-lysine. The suture is introducedthrough the CCA and advanced into the ICA and thence into the circle ofWillis (about 18-20 mm from the carotid bifurcation), effectivelyoccluding the middle cerebral artery. The silk suture around the CCA istightened around the intraluminal nylon suture to secure it andpermanently occlude the middle cerebral artery.

EXAMPLES Example 1 Impact of Gender on the Neuroprotective Efficacy ofTat-NR2B9c

The neuroprotective efficacy of Tat-NR2B9c was assessed in both male andfemale rats using the in vivo pial 3 vessel occlusion (P3VO) model ofstroke (Forder J P, Munzenmaier D H, Greene A S. Angiogenic protectionfrom focal ischemia with angiotensin II type 1 receptor blockade in therat. Am J Physiol Heart Circ Physiol 2005 April; 288(4):H1989-H1996).

Methods

Animals

Adult Sprague Dawley rats (10-12 weeks old) (males˜300 g, females˜250 g)were fasted for 12-18 hours before being subjected to permanent pialvessel occlusion of 3 terminal branches of the Middle Cerebral Arteryover the Whisker Barrel Cortex (P3VO). Tat-NR2B9c was tested in malerats plus a saline control group (n=8 in each group). Tat-NR2B9c and asaline control were tested in female rats (n=8 in each group). Theresearcher was blinded to the treatment group during the time of surgerythrough to the analysis of infarct size.

General Procedure

Rats were anesthetized with a 0.5 ml/kg intramuscular injection ofketamine (100 mg/kg), acepromazine (2 mg/kg), and xylazine (5 mg/kg),supplemented with one third the initial dose as required. An analtemperature probe was inserted and the animal was placed on a heatingpad maintained at 37° C. The right external carotid artery (ECA) wascannulated with PE 10 polyethylene tubing for dye injections. The skullwas exposed via a midline incision, scraped free of tissue, and thetemporalis muscle disconnected from the skull on the right side. Using adissecting microscope and a pneumatic dental drill, a 6×4 mm cranialwindow was made over the right somatosensory cortex (2 mm caudal and 5mm lateral to bregma) by drilling a rectangle through the skull andlifting off the piece of skull while keeping the dura intact. Afterbeing bathed with artificial cerebrospinal fluid, small boluses (10 to20 μL) of the vital dye patent blue violet (10 mmol/L; Sigma) in normalsaline, were injected into the right external carotid artery todemonstrate transit through surface vessels of the cortex. Threecritical arteriolar branches of the MCA around the barrel cortex wereselected and electrically cauterized through the dura. After thecauterizations, the bolus injections and dye transits were repeated toensure transits through the cauterized arterioles were blocked. Therectangle of skull was replaced over the window and the scalp wassutured. The catheter was removed from the ECA, the ECA was ligated, andthe anterior neck was sutured. One hour after initiation of focalocclusion, 0.3 ml of drug (3 nMol/g body weight) or saline control wereinfused through the tail vein at a rate of 0.06 ml/min. Each rat wasreturned to its individual cage under a heating lamp to maintain bodytemperature until the rat fully recovered. Food and water was suppliedad libitum.

Harvesting of Brain Tissue and Infarct Size Analysis

Twenty-four hours post-surgery, animals were re-anesthetized with 1 mLpentobarbital and the brain was quickly harvested. One coronal slice wastaken through the infarct region and incubated in 2%triphenyltetrazolium chloride (TTC) for 15 minutes at 37° C. Images werescanned and brain slices were stored at −80° C. Infarct size wasmeasured as a percent of the hemisphere for each rat in the study. Afterobtaining infarct size measurements, the animals were separated intotheir respective groups. Comparisons were made between treatment groupsas means±SE.

Results and Conclusions

The P3VO model of stroke in the rat results in a robust and reproducibleinfarct in both male and female SD rats. The Tat-NR2B9c peptide isneuroprotective in both male and female rats as seen in a significantlydecreased infarct size 24 hours after undergoing P3VO surgery (FIG. 1).Treatment with Tat-NR2B9c (3 nM/g) 1 h after stroke dramatically reducedinfarcts in animals of both genders (FIG. 1). This neuroprotectiveresponse appeared to be more pronounced in females than in males as seenby a complete lack of infarct in female rats treated with the equivalentconcentration of Tat-NR2B9c. However saline treated controls indicatethat the average infarct size in female rats is smaller (71%) than malerats.

Example 2 Peptides Containing Tat Sequence Induce Mast CellDegranulation with Histamine Release In Vitro

Methods

Cell Culture

C57 mice were sterilized with 70% ethanol, and the femur was dissectedaway from the skin and connective tissue. Bone marrow cells werecollected and resuspended in OPTI-MEM (Gibco) containing 5%heat-inactivated FBS, 6% WEHI-conditioned medium (as a source of IL-3),and 55 μM □-2mercaptoethanol. Cells were cultured at approximately 1×10⁶cells/mL. After 2 days, cells were collected and centrifuged where thepellet was plated on a fresh plate with fresh medium. New WEHI-conditionmedium was added each week. The cells were cultured for about 4 weeksafter which they were >95% mast cells and were used for the mast celldegranulation assay.

Mast Cell Degranulation Assay

Tryptase activity was determined using the Mast Cell Degranulation AssayKit (CHEMICON, Temecula, Calif.). After isolation, the cells were washedand resuspended at approximately 1×10⁶ cells/mL in 1× Assay Buffer. Fortreatment with Tat-NR2B9c or other peptides, 50 μL of solution of thefollowing concentrations: 0.125 mg/mL, 1.25 mg/mL, 12.5 mg/mL, or 125mg/mL were added to the cell suspension and 500 nM A23187 (Calcimycin),a known inducer of tryptase release in mast cells, was used as apositive control. Cells were incubated at 37° C. and 5% CO₂ for 60minutes. Cell suspension was centrifuged at 700×g and the supernatantwas carefully collected. An assay mixture (provided in the kit) wasprepared in a 96-well microtiter plate. The colorimetric reaction wasinitiated by adding 20 μL of the Tryptase Substrate to each experimentaland control well. Samples were incubated for 60 minutes at 37° C.Optical density was read at 405 nm in a microplate reader.

The following treatments were used to induce mast cell degranulation.

-   -   1) Negative control (assay buffer devoid of any peptides)    -   2) Positive Control (the calcium ionophore A23187    -   3) Tat-NR2B9c    -   4) The Tat-derived sequence of Tat-NR2B9c devoid of the PSD-95        binding sequence    -   5) NR2B9c comprising the PSD-95 binding sequence of Tat-NR2B9c        but devoid of the Tat sequence, and    -   6) AA (a 20 amino-acid peptide comprised of the Tat sequence        fused to the 9 carboxy-terminal amino acids of the NMDA NR2B        subunit, but with 2 amino acid mutations that make it incapable        of binding PSD-95).

All peptides were applied at a concentration of 125 mg/mL in order toapproximate the maximal serum concentrations attained in dogs receivinga 10 mg/kg dose of Tat-NR2B9c in some of the animal experimentsdescribed below (based on assuming a blood volume of 8% of total bodyweight).

Results and Conclusions

As seen from FIG. 2, peptides containing the Tat transduction domain allcaused mast cell degranulation, whereas the NR2B9c peptide, devoid ofthe Tat sequence, did not. In-vitro mast cell degranulation assays werecarried out in the absence of antibodies and therefore, any mast celldegranulation cannot be due to an immune phenomenon. Notably, usingRT-PCR and Western blotting, we investigated whether mast cellscontained PSD-95 protein, the therapeutic target of Tat-NR2B9c. We wereunable to detect PSD-95 in these cells (results not shown), providingfurther evidence that mast cell degranulation was unlikely to be causedby an interaction of Tat-NR2B9c with PSD-95.

In a further experiment, we determined that the degree of mast celldegranulation by Tat-NR2B9c and by the AA peptide was dose dependent.Specifically, increasing concentrations of Tat-NR2B9c evoked increasedmast cell degranulation as shown in FIG. 3.

In further experiments, we investigated the effect of sequence variationin Tat-NR2B9c on mast cell degranulation. Using the same assay, thefollowing compounds were tested (all at 50 μM):

TABLE 5 Peptide ID Peptide Name (concentration) Sequence/Structurecontrol (No peptide) CI (500 nM) Calcium IonophoreTat-NR2B9c (125 mg/ml) YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 6) 1990TAT (125 mg/ml) YGRKKRRQRRR (SEQ ID NO: 2) 1991 2B9c (125 mg/ml)KLSSIESDV (SEQ ID NO: 5) 1992 AA (125 mg/ml) YGRKKRRQRRRKLSSIEADA(SEQ ID NO: 7) 1993 F-Tat-NR2B9c (125 mg/ml) FGRKKRRQRRRKLSSIESDV(SEQ ID NO: 8) 1994 Tat-NR2B9c K > A (125 mg/m) YGRKKRRQRRRALSSIESDV(SEQ ID NO: 9) 1995 F-Tat-NR2B9c K > A (125 mg/m) FGRKKRRQRRRALSSIESDV(SEQ ID NO: 10) 1992 AA (12.5 mg/ml) YGRKKRRQRRRKLSSIEADA (SEQ ID NO: 7)

As can be seen in FIG. 4, all compounds containing Tat sequence and Tatpeptide sequence elicited mast cell degranulation, whereas NR2B9c alonedid not elicit this reaction.

Example 3 Conjugates of Peptides Containing Tat Sequence Fail to InduceMast Cell Degranulation In Vitro

The effect of certain modifications such as conjugation toTat-containing peptides on mast cell degranulation was studied usingmethods described in Example 2. The modified peptides includedTat-NR12B9c, the D-isomer of Tat-NR2B9c (termed D-Tat-NR2B9c), a biotinconjugated Tat-NR2B9c, a biotin-conjugated AMP-KLSSIESDV (SEQ ID NO:5).As shown in FIG. 5, biotin-conjugated Tat or AMP peptides to failed toinduce mast cell degranulation.

Example 4 Tat-NR2B9c Elicits Increased Histamine Levels and a HistamineResponse in Animals

Studies in Beagle Dogs

A GLP 14-day intravenous toxicity study was conducted in naïve Beagledogs (3/sex/group)(CRM Study No. 501448) in which animals received dailyinjections of 0, 0.25, 1.0, or 10 mg/kg of Tat-NR2B9c. Blood samples(approximately 1 mL) were collected from all animals on Days 1, 6 and 12at predose, 5 and 15 minutes post injection. Blood samples werecollected by venipuncture (jugular, saphenous and cephalic) into tubescontaining EDTA. The samples were then centrifuged (within 30 minutes ofcollection) in a refrigerated centrifuge (ca. 4° C.) at 2700 rpm for 10minutes. Plasma were separated into a second tube with the appropriatelabel and stored at −80° C. until analysis at CRM. Plasma samples wereused for investigating histamine levels. Samples from animals dosedintravenously with Tat-NR2B9c were analyzed using a validated method.

All animals administered 10 mg/kg Tat-NR2B9c displayed treatment-relatedclinical signs, consisting of a reddening of the muzzle, gums (alsonoted to be pale), pinna, periorbital region and limbs, and were oftenassociated with swelling. These effects were associated with lethargyand an unpalpable pulse, characterized as a severe hypotensive reactionby the attending veterinarian. These effects were observed daily,starting with the first day of dosing and persisting throughout the14-day dosing period, with no apparent adaptation by the animals. Theseeffects were not due to the development of an antibody-based immuneresponse, since these animals were not exposed to Tat-NR2B9c by thefirst day of dosing, and an increased severity of the response over the14 days of treatment was not observed. Specifically, increased histaminelevels were observed immediately following the first administration ofTat-NR2B9c to these naive Beagle dogs (see Table 6 for a summary of thedog plasma histamine levels). These animals had never been exposed toTat-NR2B9c and thus should not have memory T cells or circulatingantibodies against Tat-NR2B9c. Also, no consistent increase in histaminelevels was observed during the 14-day repeated dose toxicity study atany dose level, indicating that there is not an expansion of an antigenspecific response. Thus, the observed increases in histamine levels dueto Tat-NR2B9c are the result of direct degranulation of mast cellsrather than an antigen-specific antibody response.

TABLE 6 Determination of Histamine in Dog Plasma by Enzyme ImmunoassayDAY 12 Females Hemo- Final Assay Animal Time lyzed Dilution Result IDDate ID Point sample Factor (ng/mL) HIS-16 Feb. 12, 2006 151 Pre 1 <LLOQHIS-18 Mar. 3, 2006  5 min 1 0.204 15 min 1 0.201 HIS-10 Feb. 2, 2006152 Pre 1 <LLOQ  5 min 1 0.234 15 min 1 0.398 HIS-10 Feb. 2, 2006 153Pre 1 0.187  5 min H 1 0.546 15 min 1 0.513 HIS-10 Feb. 2, 2006 154 Pre1 0.184  5 min 1 0.392 15 min 1 0.207 HIS-10 Feb. 2, 2006 155 Pre 1<LLOQ  5 min 1 0.609 15 min 1 3.339 HIS-10 Feb. 2, 2006 156 Pre 1 <LLOQ 5 min 1 0.190 15 min 1 <LLOQ HIS-10 Feb. 2, 2006 251 Pre 1 <LLOQ HIS-16Feb. 12, 2006  5 min 1 <LLOQ 15 min 1 0.273 HIS-10 Feb. 2, 2006 252 Pre1 <LLOQ HIS-11 Feb. 3, 2006  5 min 1 0.252 15 min 1 0.193 HIS-11 Feb. 3,2006 253 Pre 1 <LLOQ  5 min 1 0.213 15 min 1 0.293  5 min H 1 0.912 15min 1 0.196 HIS-11 Feb. 3, 2006 353 Pre 1 0.385  5 min H 1 0.282 15 min1 0.446 HIS-12 Feb. 6, 2006 451 Pre 1 <LLOQ  5 min H 3 1.642 HIS-17 Mar.3, 2006 15 min 1 <LLOQ HIS-12 Feb. 6, 2006 452 Pre 1 0.188  5 min H 36.154 15 min H 3 0.565 HIS-12 Feb. 6, 2006 453 Pre h 1 0.302  5 min 313.937  HIS-17 Mar. 3, 2006 15 min h 1 0.587 HIS-12 Feb. 6, 2006 454 Pre1 <LLOQ HIS-17 Mar. 3, 2006  5 min 1 0.504 15 min 1 0.312 HIS-12 Feb. 6,2006 455 Pre h 1 <LLOQ  5 min h 3 2.335 HIS-17 Mar. 3, 2006 15 min 10.312 HIS-18 Mar. 3, 2006 456 Pre 1 0.351  5 min h 1 0.485 HIS-16 Feb.12, 2006 15 min 1 0.330 LLOQ = 0.180 ng/mL h = sample was hemolyzedCardiovascular Effects of Tat-NR2B9c Indicative of Histamine Release inDogs

In a GLP cardiovascular telemetry study in unrestrained conscious Beagledogs (CRM Study No. 691106), 6 animals (3 males, 3 females) wereadministered escalating doses of Tat-NR2B9c (0.25, 1.0, or 5.0 mg/kggross peptide), with a washout period of 3 days between dose levels. Noeffects on blood pressure were observed at 0.25 or 1.0 mg/kg. Atransient drop in blood pressure was observed in 4 of 6 dogs at 5 mg/kg,lasting approximately 30 minutes. The finding that a drop in bloodpressure may have been dose-related indicates that the drop was not dueto an allergic (antibody mediated) immune response. Moreover, thetime-frame between the low dose (0.25 mg/kg) and the high dose (5.0mg/kg) was about 6 days, i.e., of insufficient duration to allow thegeneration of an immune response. The effect is thus caused by a directdegranulation of mast cells causing histamine release.

To obtain detailed cardiovascular information at the highest dose leveltested in the 14-day dog repeated-dose toxicity study (i.e., 10 mg/kg),an additional GLP cardiovascular telemetry study in unrestrained,conscious, Beagle dogs was performed (CRM Study No. 691429). Six animals(3 males, 3 females) received vehicle in the morning and 10 mg/kgTat-NR2B9c in the afternoon of the same day (at least 4 hours betweendoses). Increases in heart rate were observed for up to 15 minutespost-dose in treated animals, with the maximum effect observed at 10minutes in males and females. Decreases in blood pressure values (up to62%) were observed in individual animals at 5 to 10 minutes post-dose.The female animals used in this additional cardiovascular study wereobtained from the Charles River colony and were non-naïve animals thathad been previously used in the first cardiovascular study forTat-NR2B9c (CRM Study No. 691106). The effects observed at 10 mg/kg inCRM Study No. 691429 were comparable to the clinical signs observed inthe 14-day dog toxicity study (CRM Study No. CRM Study No. 501448), withmore severe blood pressure effects observed for treated animals thanthat observed at the highest dose level (5 mg/kg) tested in CRM StudyNo. 691106. These results again indicate a direct degranulation of mastcells.

We conducted non-GLP studies of the effects of Tat-NR2B9c on bloodpressure in anesthetized rats receiving 50 mg/kg of Tat-NR2B9c. Thisdose was selected for the rat as it produced decreased tidal volume,respiratory rate and derived minute volume. In one experiment, 5 maleSprague-Dawley rats received a 50 mg/kg Tat-NR2B9c bolus dose over 3minutes. Blood pressure was monitored via a femora arterial catheter.

All animals experienced transient reductions in mean arterial pressureas shown in FIG. 6. Another experiment, in which 6 animals weresimilarly tested, showed similar results. As discussed above in the caseof dogs, these reactions in rats were also observed in naïve animalsthat had not had any prior opportunity to develop an immune response toTat-NR2B9c. These data provide evidence in a second species of mast celldegranulation by peptides containing Tat sequence.

Inflammatory Reactions Indicative of Histamine Release in Dogs

A non-GLP study was conducted to examine a dose range for Tat-NR2B9c,administered to Beagle dogs by a single, slow intravenous injection. Twoanimals (one male and one female Beagle dog) were dosed intravenouslywith Tat-NR2B9c on seven occasions. There was a 3-4 day wash-out periodbetween the doses. The first dose was given at 2.5 mg/kg. Since theanimals did not show any signs of toxicity, the second dose wasadministered at 7.5 mg/kg. The male animal displayed angio-neuroticedema of the soft tissue of the head and urticaria type of reaction,especially on the ventral aspect of the abdomen. There was no reactionin the female dog. Vital signs (heart rate, blood pressure, respiratoryrate and body temperature) stayed within the normal physiological rangesin both animals. The third dose was given at 12.5 mg/kg. After dosing,angio-neurotic oedema and urticaria were observed in both animals. Thereaction in the male dog was assessed to be moderate, and in the femaleanimal, the reaction was mild. The next dose was set at 20.0 mg/kg.After dosing, the male animal went into shock, where blood pressure (BP)and pulse were undetectable. The animal was treated with i.v.administration of benadryl and dexamethasone. BP at 5 minutespost-dosing was recorded as 37/13 mm Hg (normal BP in a dog is ˜160/90).A decision was made not to dose the female animal.

The next doses were given in order to better understand the type ofreactions seen in preceding doses. The fifth and sixth doses were set at2.5 and 5.0 mg/kg. At 2.5 mg/kg with the exception of reddening of earand face of the male dog, no other adverse reactions were observed ineither dog. At 5.0 mg/kg, a moderate reaction was seen in the maleanimal, while there was no reaction in the female dog.

It was concluded that Tat-NR2B9c at high doses is capable of inducingprofound transient hypotension and urticaria-like skin reactions. Thesereactions appeared to be dose dependent, and the male animal appeared tobe more sensitive to the test article than the female animal.

Example 5 Treatment with Antihistamine Prevents Symptoms Induced byTat-NR2B9c in Dogs

Both animals from Example 4 were next administered 12.5 mg/kg ofTat-NR2B9c after pre-treatment with benadryl at 1 mg/kg administered 30minutes before Tat-NR2B9c. There was slight reddening of inner skin ofthe ears in the male animal. The male animal also vomited ˜15-20 minutesafter the administration of Tat-NR2B9c. There was no reaction observedin the female dog. Accordingly, pretreatment with the antihistamine drugbenadryl prevented the angio-neurotic oedema and urticaria reactionsthat were earlier observed in both animals at the same dose level ofTat-NR2B9c. The results indicate that antihistamines such as benadryl,and of corticosteroids such as dexamethasone effectively treat theadverse consequences of mast cell degranulation.

Taken together, these results provide direct experimental evidence thatadministration of Tat-NR2B9c elicits an elevation in blood histaminelevels in experimental animals, that increased histamine levels are dueto mast cell degranulation, and that treating this response withantihistamine medications and with corticosteroids may constitute andeffective means by which to administer Tat-NR2B9c and other compoundscontaining protein translocation domains such as Tat.

Example 6 Direct Evidence that Tat-NR2B9c Elicits Blood HistamineElevations in Humans

Methods

We carried out a Safety, Tolerability and Pharmacokinetic Study ofTat-NR2B9c in humans. Subjects were either normal, healthy, non-smokingmales or post-menopausal or surgically sterile female subjects with aminimum age of 18 years. The subjects were either administeredTat-NR2B9c, Lot #: 124-134-001B, or were given placebo (PhosphateBuffered Saline), Lot #: 124-134-001A, administered as an intravenousinfusion (10±1 minutes). Four subjects were dosed in each of Cohorts 1to 3, and 10 subjects were dosed in each of Cohorts 4 to 8. All 62subjects completed the study. Treatment periods for each cohort were asfollows: Cohort 1: Sep. 14, 2006; Cohort 2: Sep. 26, 2006; Cohort 3:Oct. 6, 2006; Cohort 4: Oct. 20, 2006; Cohort 5: Nov. 6, 2006; Cohort 6:Dec. 4, 2006; Cohort 7: Dec. 17, 2006; Cohort 8: Feb. 25, 2007.

Blood Draw Timepoints:

During the study period, 11 blood samples were collected forpharmacokinetic analysis from each subject at the following timepoints:0.00 (pre-dose), 0.08 (5 minutes), 0.17 to 0.25 (10 to 15 minutes,precisely at the end of each individual drug infusion), 0.33 (20minutes), 0.50, 0.75, 1.00, 2.00, 6.00, 12.00, and 24.00 hourspost-dose. In addition, 8 blood samples were collected for histamineanalysis from each subject at the following timepoints: 0.00 (pre-dose),and at 0.08 (5 minutes), 0.17 (10 minutes), 0.25, 0.50, 1.00, 2.00, and24.00 hours post-dose.

Safety Assessment:

The safety assessment was performed on all subjects who received atleast 1 dose during the course of the study. The incidents of alladverse events (AEs) were tabulated by treatment and subject number.Absolute values for vital signs, electrocardiogram (ECG) parameters,laboratory parameters and physical examinations were also documented andvalues outside the normal range were flagged. Shifts from baselinevalues were tabulated. AEs were documented using investigator andMedical Dictionary for Regulatory Activities (MedDRA) terms.

Results

Part 1: Effects of Tat-NR2B9c on Blood Histamine Levels:

A summary of abnormal histamine results by dose is illustrated in Table7. Seven of 8 subjects in the 3.75 mg/kg dose group had histamine levelsgreater than 10 nmol/L (average 24.3 nmol/L; maximum of 39.8 nmol/L) 10minutes after the start of NA 1 administration, and 3 of the subjectsstill had histamine levels greater than 10 nmol/L (average 15.3 nmol/L;maximum of 20.3 nmol/L) 15 minutes after the start of NA 1administration.

Other than the 3.75 mg/kg dose group, no treatment group had significantabnormal levels of histamine. The placebo group and the 0.375 mg/kg dosegroup each had 1 subject that had an elevated histamine level at 1timepoint, but these results were at screening and at 2.00 hours postdose, respectively. All abnormal histamine results returned to thenormal range within 24.00 hours of drug administration.

TABLE 7 Number of Subjects with Histamine levels >10 nmol/L by TreatmentGroup Number of Subjects Dose of NA-1 (mg/kg) Time Placebo 0.02 0.080.20 0.375 0.75 1.50 2.60 3.75 Day (hr) (n = 16) (n = 2) (n = 2) (n = 2)(n = 8) (n = 8) (n = 8) (n = 8) (n = 8) Screening N/AP 0 0 0 0 1 0 0 0 0 1 0.00 0 0 0 0 0 0 0 0 0 0.08 0 0 0 0 0 0 0 0 0 0.17 0 0 0 0 0 0 0 0 70.25 0 0 0 0 0 0 0 0 3 0.50 0 0 0 0 0 0 0 0 0 1.00 0 0 0 0 0 0 0 0 02.00 1 0 0 0 0 0 0 0 0 24.00  0 0 0 0 0 0 0 0 0  7 N/AP 0 0 0 0 0 0 0 00 14 N/AP 0 0 0 0 0 0 0 0 0 28 N/AP 0 0 0 0 0 0 0 0 0 End- of- studyPart 11: Safety Data

Forty subjects who participated in the study experienced a total of 168adverse effects (AEs) during the study. The majority of AEs were mild inseverity. Thirty-four of 46 active treatment subjects (73.9%)experienced at least 1 AE, while 6 of 16 placebo treatment subjects(37.5%) experienced at least 1 AE. Subjects in the 2.60 and 3.75 mg/kgdose groups experienced significantly more AEs than subjects in thelower dose groups. No Serious Adverse Events (SAEs) were reported. Themost common AEs experienced by subjects receiving Tat-NR2B9c werefeeling hot ( 13/46; 28.3%), pruritis ( 12/46; 26.1%), flushing ( 10/46;21.7%), and dry mouth ( 9/46; 19.6%). All AEs were resolved with theexception of 2 instances of increased blood glucose, as the subjectswere lost to follow-up.

The incidence of AEs in the 2.60 and 3.75 mg/kg dose groups was higherthan the AE incidence rate in the placebo, 0.02, 0.08, 0.20, 0.375, 0.75and 1.50 mg/kg dose groups. At doses of Tat-NR2B9c≧22.60 mg/kg, severalAEs were frequently reported. These included: (1) decreases in bloodpressure, (2) tingling sensation (paraesthesia), (3) numbness(hypoaesthesia), (4) redness (erythema), (5) rash, (6) itchiness(pruritus), (7) dry mouth, (8) nausea, (9) feeling hot, and (10)flushing. The onset of these AEs coincided with the administration ofthe study drug and was probably related to the study drug.

In preclinical trials with Tat-NR2B9c, elevated histamine levels wereobserved in high dose groups, and were likely the source of side effectsincluding swelling, redness and hypotension. In the current study,histamine levels were elevated in 7 of the 8 subjects in the highestdose group (3.75 mg/kg) 10 minutes after the start of the intravenousdrug administration, and remained elevated in 3 of these subjects 15minutes after drug administration, after which time levels returned tothe normal range. During the same time frame that histamine levels wereelevated, most of the AEs in the 3.75 mg/kg dose group were observed.This suggests that the elevated histamine levels were the source of themost frequently reported AEs (including decreased blood pressure,tingling, numbness, redness, rash, itchiness, dry mouth, nausea, feelinghot, and flushing).

Most of the listed AEs were also observed in preclinical animal trialswhere the Maximum Tolerated Dose (MTD) was established at 12.5 and 100mg/kg for dogs and rats, respectively. Most of the AEs in the 2.60 and3.75 mg/kg dose groups were not observed, or observed in only 1 subjectin the dose groups between 0.02 and 1.50 mg/kg. This suggests that theAEs that were observed at higher doses of Tat-NR2B9c were minimal or notpresent at this lower dose range.

Example 7

Materials

TAT-NR2B9c chemically synthesized by AnaSpec Inc (San Jose, Calif.).Rv-Tat-NR2B9c chemically synthesized by Sickkids Hospital AdvancedProtein Technology Centre (Toronto, ON, Canada). All peptides werehigh-performance liquid chromatography purified to >95%. Peptide stocks(3 mM) were prepared in sterile saline and stored in aliquots at −80° C.Cromolyn, pyrilamine, ranitidine, oxatomide and Dexamethasone purchasedfrom Sigma-Aldrich (St. Louis, Mo.)

Rat were put to sleep in a chamber with 2% isoflurane and a gas of 2 Loxygen, and then transferred to a face mask with reduced gas (1%isoflurane and a gas of 2 L oxygen) once asleep. Femoral cut down ofartery and vein for catheterization with PE50 tubing. These arteries andveins provide access for continual monitoring of mean arterial pressure(MAP) and for drug infusion respectively. Cromolyn (1 mg/kg/min) orsaline (1 ml/kg/min) was infused at for 5 minutes then TAT-NR2B9c,Rv-Tat-2B9c (3 μM/kg in saline) or saline (1 ml/kg) was give by bolusinjections immediately after saline or cromolyn infusion. Other drugsusing these studies were infused 10 minutes before TAT-NR2B9c injection.Surgeries were done on animal's the left side. Animal's blood pressure(HEWLETT PACKARD Blood pressure system, model 78304A) and bodytemperature (Digi Sense Thermometer, model 8528-10) was monitored every1 minute within 60 minutes. Saline (1 ml/kg) was given to all groups atbaseline, and the control group received a further 2 ml/kg saline ateach time point. Results presented are the average MAP from 5 rats. Asummary of all experiments performed (n=10 each drug) is presented inthe respective graphs as mean±SEM, (Student's test, *, P<0.05 and ***,p<0.001)

FIG. 7 shows that both Tat-NR2B9c and Rv-Tat-NR2B9 (tat attached toNR2B9c in reverse orientation) at the high dose of 7.5 mg/kg give arapid and transient reduction in MAP over a period of about 0-6 minafter injection.

FIGS. 8A-D show the effect of 5 mg/kg cromolyn administeredintravenously about five minutes before the administration ofTat-NR2B9c. FIG. 8A shows a time course of MAP for treatment withTat-NR2B9c alone, treatment with Tat-NR2B9c plus cromolyn or treatmentwith cromolyn, plus saline as a control. FIG. 8B shows areas under thecurve. FIG. 8C shows minimal MAP value. FIG. 8D shows maximum percentagedecline in MAP. Asterisks indicated a statistically significant result.FIG. 8B shows that treatment with cromolyn significantly reduces thedecline in MAP due to Tat-NR2B9c. Cromolyn did not itself affect MAP inthe absence of Tat-NR2B9c as shown by the cromolyn saline control. Thecomparisons shown in FIGS. 8B-D further illustrate the significanteffect of cromolyn in inhibiting decline of blood pressure.

The same experiment was performed with Rv-Tat-NR2B9c used in place ofTat-NR2B9c and similar results were obtained as shown in FIGS. 8E and F.That is, cromolyn significantly inhibited decline of MAP values due toRv-Tat-NR2B9c.

FIGS. 9A-D present similar data except that diphenhydramine (12.5 mg/kg)(Benadryl), a histamine H1 antagonist, was used as an anti-inflammatoryagent in place of cromolyn. Diphenhydramine also significantly inhibiteddecline in blood pressure as shown in FIGS. 9A, B and D. However,administration of diphenhydramine itself caused a sharp reduction inblood pressure before Tat-NR2B9c was administered. The experiment wasrepeated except that diphendydramine was used at 1 mg/kg.Diphendydramine was not observed to have a significant effect oninhibiting decline of blood pressure due to Tat-NR2b9c at this dosage.Diphenhydramine also did not itself reduce blood pressure at thisdosage.

The experiment was repeated with another H1 antagonist pyrilamine.Although the maximal decline in blood pressure due to Tat-NR2B9c wasslightly reduced, the reduction did not achieve statistical significance(see FIGS. 10 A-D).

FIG. 11A presents similar data for a combination of diphenhydramine(12.5 mg/kg) and H2 antagonist Ranitidine (10 mg). The combined agentsthemselves lowered MAP (presumably due to the effect of diphenhydramineand inhibited reduction due to Tat-NR2B9c. The inhibition of reductionwas significant as shown by the analyses in FIGS. 11B-D. The experimentwas repeated using only Ranitidine. Ranitidine had no effect on MAPitself and any effect in inhibiting decline of MAP due to Tat-NR2B9c wasslight and not statistically significant.

A similar experiment was performed with 6 mg/kg dexamethasone as theanti-inflammatory agent. Dexamethasone was administered about ten minbefore TAT-NR9B9c. Dexamethasone was not observed to have a significanteffect on inhibiting decline in blood pressure due to Tat-NR2B9c in thisexperiment.

A similar experiment was performed with 6 mg/kg lodoxamide co-formulatedwith 3 mg/kg Tat-NR2B9c compared with 3 mg/kg Tat-NR2B9c. Solution madefresh by combining 1.89 ml 20 mg/ml Tat-NR2B9c in 0.9% saline with 3.11ml 0.1% Alomide® (lodoxamide) and vortexing. Each mL of ALOMIDE®contains: 1.78 mg lodoxamide tromethamine equivalent to 1 mg lodoxamide,preservative benzalkonium chloride 0.007%, mannitol, hydroxypropylmethylcellulose 2910, sodium citrate, citric acid, edetate disodium,tyloxapol, hydrochloric acid and/or sodium hydroxide (to adjust pH), andpurified water.

The co-formulation was stable. By contrast, a co-formulation ofTat-NR2B9c with cromolyn tended to precipitate. Animals (male, SpragueDawley) were allowed to eat and drink before surgery. Animals were putto sleep in a chamber with 2% isoflurane and 1 L oxygen. Once asleep,rat was transferred to a face mask at 1% isoflurane and a gas of 1 Loxygen. PE50 tubing was used for connection of the femoral artery andvein. These arteries and veins provide access for continual monitoringof arterial pressure and for drug infusion (50 mg per kg in a volume of2 ml per kg injected with in 2 minutes). Surgeries were done on the leftside. Animal's blood pressure and body temperature was monitored for 90minutes. FIG. 13A shows a MAP timecourse following either a 2 minuteinfusion of 3 mM/kg Tat-NR2B9c with 6 mg/kg Lodoxamide (Mast cellstabilizer) or 3 mM/kg Tat-NR2B9c alone as a control in a 2 ml volume.Co-treatment with lodoxamide can completely abrogate the drop in MAPresulting from Tat-NR2B9c injection. Cromolyn infused for three minutesimmediately before administration of TAT2B9c also completely abrogatedthe drop in MAP (FIG. 13B).

Example 8

Rv-Tat-NR2B9c was compared with Tat-NR2B9c in a model of ischemia.Rv-Tat-NR2B9c is the same as Tat-NR2B9c except that the order of aminoacids from N—C in the tat portion of the peptide is reversed inRv-Tat-NR2B9c.

Methods for Three pial vessel occlusion model of ischemia (3PVO):Experiments were performed on fasted rats (free overnight access towater but not food). Permanent three pial vessels occlusion (3PVO) wasperformed as described by Forder et al., Am. J. Physiol. Heart Circ.Physiol. 2005 April; 288(4):H1989-96. In brief, rats were anesthetizedwith a 0.5 ml/kg intramuscular injection of ketamine (100 mg/kg),acepromazine (2 mg/kg), and xylazine (5 mg/kg), supplemented withone-third of the initial dose as required. A rectal temperature wasmonitored and the animal body temperature was maintained at 37° C. byusing a heating pad. The skull was exposed via a midline incision andscraped free of tissue. Using a dissecting microscope and a pneumaticdental drill, a 6- to 8-mm rectangular cranial window was made over theright somatosensory cortex (2 mm caudal and 5 mm lateral to bregma) andthe loose piece of skull was removed while keeping the dura intact. The3 pial arteriolar middle cerebral artery branches were electricallycauterized around the barrel cortex area. Incision was sutured with 3.0silk sutures. Animals were returned to individual cages under a heatinglamp to maintain body temperature until the rats fully recovered. Foodand water was supplied. One hour after 3PVO ischemia the rats wereinjected with 3 μM/kg Tat-NR2B9c or Rv-Tat-NR2B9c intravenously throughtail vein. Twenty-four hours after surgery, the brain was quicklyharvested, sliced (2 mm thick) and incubated in 2% triphenyltetrazoliumchloride (TTC) (Sigma-Aldrich, St. Louis, Mo.) in saline for 15 min at37° C. Images were scanned (CanoScan, 4200F, Canon). Infarct percentagewas calculated per slice using Image J software (NIH).

Rv-Tat-NR2B9c is as or more effective than Tat-NR2B9c at reducinginfarcts in the 3PVO model. FIG. 14 shows a bar graph showing theaverage infarct size for each group.

Example 9

A co-formulation of lodoxamide and Tat-NR2B9c as described above wastested in comparison with Tat-NR2B9c and vehicle controls was tested ona rat 3PVO model of stroke as described above. The area of resultinginfarctions is shown in FIG. 15. Tat-NR2B9c significantly inhibitedinfarction size relative to vehicle control. However, surprisingly thelodoxamide Tat-NR2B9c combination resulted in a statisticallysignificant reduction relative to Tat-NR2B9c alone. Thus, peripheralco-administration of lodoxamide not only reduces inflammation due toTat-NR2B9c but also increases its efficacy in reducing infarcts.

Although the invention has been described in detail for purposes ofclarity of understanding, it will be obvious that certain modificationsmay be practiced within the scope of the appended claims. Allpublications and patent documents cited in this application are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted. To the extent differencesequences might be associated with the same accession number atdifferent times, the sequence associated with the accession number atthe effective filing date is meant. The effective filing date means theearliest priority date at which the accession number at issue isdisclosed. Unless otherwise apparent from the context any element,embodiment, step, feature or aspect of the invention can be performed incombination with any other.

What is claimed is:
 1. A method of delivering a pharmacological agentlinked to an internalization peptide comprising RKKRRQRRR (SEQ ID NO:51)to a subject having or a risk of a disease treatable by thepharmacological agent, comprising administering the pharmacologicalagent linked to the internalization peptide to the subject, andadministering a mast cell degranulation inhibitor and/or ananti-histamine thereby reducing an inflammatory response due to mastcell degranulation induced by the internalization peptide, wherein theinflammatory response occurs within 60 minutes of administering thepharmacologic agent linked to the internalization peptide.
 2. The methodof claim 1, wherein the anti-inflammatory agent is administered within awindow of 15 minutes before to the same time as administering thepharmacologic agent.
 3. The method of claim 1, wherein the mast celldegranulation inhibitor and the antihistamine are administered.
 4. Amethod of delivering a pharmacologic agent to a subject, the methodcomprising: administering the pharmacologic agent linked to aninternalization peptide having an amino acid sequence comprisingRKKRRQRRR (SEQ ID NO:51) to the subject; and administering ananti-inflammatory agent to the subject, whereby the anti-inflammatoryagent inhibits an inflammatory response from mast cell degranulationinduced by the internalization peptide, wherein the inflammatoryresponse occurs within 60 minutes of administering the pharmacologicagent linked to the internalization peptide.
 5. The method of claim 4,wherein the anti-inflammatory agent is a mast cell degranulationinhibitor.
 6. The method of claim 5, wherein the anti-inflammatory agentis administered at the same time as or up to 15 minutes before thepharmacological agent.
 7. The method of claim 5, wherein theanti-inflammatory agent is co-formulated with the pharmacologic agent.8. The method of claim 5, for treating or prophylaxis of a diseasemediated by excitotoxicity in the subject.
 9. The method of claim 8,wherein the pharmacological agent is PL peptide of an NMDAR receptor.10. The method of claim 9, wherein the internalization peptide is a tatpeptide.
 11. The method of claim 10, wherein the internalization peptidehas an amino acid sequence comprising GRKKRRQRRR (SEQ ID NO:1),YGRKKRRQRRR (SEQ ID NO:2), FGRKKRRQRRR (SEQ ID NO:3) or GRKKRRQRRRPQ(SEQ ID NO:4) or RRRQRRKKRGY (amino acids 1-11 of SEQ ID NO:70).
 12. Themethod of claim 8, wherein the disease is stroke.
 13. The method ofclaim 8, wherein the subject is at risk of transient cerebral ischemicattack as a result of undergoing surgery.
 14. The method of claim 5,wherein the mast cell degranulation inhibitor is cromolyn, nedocrimil,tranilast, lodoxamide, azelastine, bepotastine, chlorzoxazone,epinastine, isoproterenol, olopatadine, pemirolast, pimecrolimus orpirbuterol.
 15. The method of claim 4, wherein the anti-inflammatoryagent is administered by a peripheral route.
 16. The method of claim 15,wherein the subject is suffering from an episode of a disease and thepharmacological agent and the anti-inflammatory agent are administeredonce during the disease episode.
 17. The method of claim 15, wherein theadministration of the anti-inflammatory agent does not comport with arecurring regime of administering the anti-inflammatory agent to thepatient without the pharmacologic agent.
 18. The method of claim 4,wherein the anti-inflammatory agent is administered within a window of30 minutes before to 15 after administering the pharmacologic agent. 19.The method of claim 4, wherein the anti-inflammatory agent is a mastcell degranulation inhibitor or anti-histamine and the anti-inflammatoryagent and pharmacological agent linked to the internalization peptideare co-formulated.
 20. The method of claim 19, wherein theco-formulation is administered intravenously.
 21. The method of claim 4,wherein the anti-inflammatory agent is an anti-histamine.
 22. The methodof claim 21, wherein the anti-inflammatory agent is azatadine,azelastine, burfroline, cetirizine, cyproheptadine, doxantrozole,etodroxizine, forskolin, hydroxyzine, ketotifen, oxatomide, pizotifen,proxicromil, an N,N′-substituted piperazine, erfenadine, acrivastine,astemizole, cetirizine, loratadine, mizolastine, levocetirizine,desloratadine, fexofenadine, ketotifen fumarate, mequitazine,dexbrompheniramine, brompheniramine methdilazine, chlorpheniramine,terbutaline, pimecrolimus or diphenhydramine.
 23. The method of claim21, wherein the anti-inflammatory agent is an H1 antagonist.
 24. Themethod of claim 21, wherein the anti-inflammatory agent is an H1antagonist administered with an H2 antagonist.
 25. The method of claim24, wherein the H1 antagonists is diphenhydramine and the H2 antagonistis ranitidine.
 26. The method of claim 4, wherein the anti-inflammatoryagent is an anti-histamine or mast cell degranulation inhibitor and thepharmacological agent and the anti-inflammatory agent are administeredintravenously.
 27. The method of claim 4, wherein the pharmacologicalagent is administered at a dose greater or equal to 2.6 mg/kg when thepharmacological agent is TAT-NR2B9c or equivalent dose to deliver anequimolar amount of internalization peptide in another pharmacologicalagent.
 28. The method of claim 4, wherein the pharmacological agent andanti-inflammatory agent are administered by overlapping or coextensiveintravenous infusion.
 29. The method of claim 28, wherein thepharmacological agent mast cell degranulation inhibitor andantihistamine are administered intravenously.